Microarray having a base cleavable linker

ABSTRACT

There is disclosed a microarray having base cleavable linkers and a process of making the microarray. The microarray has a solid surface with known locations, each having reactive hydroxyl groups. The density of the known locations is greater than approximately 100 locations per square centimeter. Optionally, oligomers are synthesized in situ onto the cleavable linkers and subsequently cleaved using a cleaving base. Optionally, the oligomers are cleaved and recovered as a pool of oligomers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of U.S. patent applicationSer. No. 11/229,757, filed Sep. 19, 2005 now abandoned and acontinuation of U.S. patent application Ser. No. 11/361,160, filed Feb.24, 2006, now U.S. Pat. No. 7,541,314, both of which applications arehereby incorporated by reference in their entirety for all purposes.

TECHNICAL FIELD

Disclosed herein are microarrays having base cleavable linker moietiesattached at known locations on the microarray. Further disclosed is aprocess to make the microarrays having the base cleavable linkersattached at known locations. Further disclosed are oligomers synthesizedin situ onto the base cleavable linkers. Further disclosed is cleavingthe oligomers from the microarray to provide a pool of oligomers.

BACKGROUND

Microarray preparation methods for synthetic oligomers, includingoligonucleotides (oligos) include the following: (i) spotting a solutionon a prepared flat or substantially planar surface using spottingrobots; (ii) in situ synthesis by printing reagents via ink jet or othercomputer printing technology and using standard phosphoramiditechemistry; (iii) in situ parallel synthesis using electrochemicallygenerated acid for removal of protecting groups and using standardphosphoramidite chemistry; (iv) in situ synthesis using masklessphoto-generated acid for removal of protecting groups and using regularphosphoramidite chemistry; (v) mask-directed in situ parallel synthesisusing photo-cleavage of photolabile protecting groups (PLPG) andstandard phosphoramidite chemistry; (vi) maskless in situ parallelsynthesis using PLPG and digital photolithography and standardphosphoramidite chemistry; and (vii) electric field attraction/repulsionfor depositing fully formed oligos onto known locations.

Photolithographic techniques for in situ oligo synthesis are disclosedin Fodor et al. U.S. Pat. No. 5,445,934 and the additional patentsclaiming priority thereto, all of which are incorporated by referenceherein. Electric field attraction/repulsion microarrays are disclosed inHollis et al. U.S. Pat. No. 5,653,939 and Heller et al. U.S. Pat. No.5,929,208, both of which are incorporated by reference herein. Anelectrode microarray for in situ oligo synthesis using electrochemicaldeblocking is disclosed in Montgomery U.S. Pat. Nos. 6,093,302;6,280,595, and 6,444,111 (Montgomery I, II, and III respectively), allof which are incorporated by reference herein. Another and materiallydifferent electrode array (not a microarray) for in situ oligo synthesison surfaces separate and apart from electrodes using electrochemicaldeblocking is disclosed in Southern U.S. Pat. No. 5,667,667, which isincorporated by reference herein. A review of oligo microarray synthesisis provided by: Gao et al., Biopolymers 2004, 73:579.

U.S. patent application Ser. No. 10/243,367, filed 12 Sep. 2002(Oleinikov) discloses a process for assembling a polynucleotide from aplurality of oligonucleotides. The claimed process provides a pluralityof oligonucleotide sequences that are synthesized in situ or spotted ona microarray device. The plurality of oligonucleotide sequences isattached to a solid or porous surface of the microarray device. Theoligonucleotide sequences are cleaved at a cleavable linker site to forma soluble mixture of oligonucleotides. The cleavable linker is achemical composition having a succinate moiety bound to a nucleotidemoiety such that cleavage produces a 3′-hydroxy nucleotide.

The succinate moiety disclosed in Oleinikov as a cleavable linker isbound to the solid or porous surface through an ester linkage byreacting the succinate moieties with the solid or porous surface. Ingeneral, formation of an ester linkage to an organic hydroxyl on a solidsurface using a succinate is relatively difficult and often results inrelatively low yield. Additionally, the reaction conditions require arelatively long period of time at relatively high temperature.Increasing yield would increase oligonucleotide density and provide moreefficient production of oligonucleotides on a microarray.Oligonucleotides cleaved from the microarray disclosed in Oleinikov havea three prime hydroxyl, which may limit the use of such oligonucleotidesor result in the need for an additional step to modify the three-primehydroxyl. Disclosed herein are embodiments that provide oligonucleotideshaving three-prime functionality that is different from a three primehydroxyl of Oleinikov, of which different functionality expands the useof such oligonucleotides. Further disclosed herein are embodiments thataddress the problems in Oleinikov of low yield and hence lowoligonucleotide density at a location on a microarray by providingalternative cleavable linker chemistry, which is more reactive tohydroxyl groups on a microarray. Further disclosed herein areembodiments that address the limitations of Oleinikov with respect toattachment of oligonucleotides having a three-prime hydroxyl to a solidor porous surface by providing different three-prime chemistry.

SUMMARY

Disclosed herein is a process for forming a microarray having basecleavable sulfonyl linkers. The process comprises providing an arrayhaving known locations having a plurality of hydroxyl groups. The arraycomprises a surface or a matrix proximate to the surface, wherein thedensity of the known locations is greater than approximately 100 persquare centimeter. The process further comprises bonding one or aplurality of sulfonyl amidite containing reagents to the hydroxyl groupsat the known locations to form a plurality of cleavable linkers bondedto the known locations. The cleavable linkers comprise a hydroxyl moietyand a base-labile cleaving moiety. A phosphorous-oxygen bond is formedbetween phosphorous of the sulfonyl amidite containing reagent andoxygen of the hydroxyl groups.

Further disclosed herein is a process for forming a microarray havingbase cleavable sulfonyl linkers. The process comprises providing anarray device having a plurality of known locations, each having aplurality of hydroxyl groups. The density of the known locations isgreater than approximately 100 per square centimeter. The processfurther comprises bonding a plurality of sulfonyl amidite moieties tothe hydroxyl groups to form a plurality of cleavable linkers attached tothe array device at each known location. The cleavable linkers comprisea linker hydroxyl moiety and a base-labile cleaving moiety. Aphosphorous-oxygen bond is formed between phosphorous of the sulfonylamidite moieties and oxygen of the hydroxyl groups. The process furthercomprises synthesizing a plurality of oligomers onto the linker hydroxylmoieties.

Further disclosed herein is a process for forming a microarray havingbase cleavable sulfonyl linkers. The process comprises providing anarray device having a plurality of known locations, each having aplurality of hydroxyl groups. The density of the plurality of knownlocations is greater than approximately 100 per square centimeter. Theprocess further comprises bonding a plurality of sulfonyl amiditemoieties to the hydroxyl groups to form a plurality of cleavable linkersbonded to the known locations. The cleavable linkers comprise a linkerhydroxyl moiety and a base-labile cleaving moiety. A phosphorous-oxygenbond is formed between phosphorous of the sulfonyl amidite moieties andoxygen of the hydroxyl groups. The process further comprisessynthesizing a plurality of oligomers covalently bound to the linkerhydroxyl moiety. The process further comprises cleaving the oligomersfrom the known locations at the base-labile cleaving moiety using acleaving base. The oligomers are recoverable. The oligomers comprisingDNA and RNA have a 3′ phosphate after cleaving from the solid surface.

Further disclosed herein is a process for forming a pool of oligomersproduced by providing an array having known locations having a pluralityof hydroxyl groups. The array comprises a surface or a matrix proximateto the surface. The density of the known locations is greater thanapproximately 100 per square centimeter. The process further comprisesbonding a plurality of sulfonyl amidite moieties to the hydroxyl groupsto form a plurality of cleavable linkers bonded to the known locations.The cleavable linkers comprise a linker hydroxyl moiety and abase-labile cleaving moiety. A phosphorous-oxygen bond is formed betweenphosphorous of the sulfonyl amidite moieties and oxygen of the hydroxylgroups. The process further comprises synthesizing a plurality ofoligomers covalently bound to the linker hydroxyl moiety. The processfurther comprises cleaving the oligomers from the known locations at thebase-labile cleaving moiety using a cleaving base. The oligomerscomprise DNA and RNA and have a 3′ phosphate after cleaving from thesolid surface. The oligomers are oligonucleotides having a 3′ phosphate.The pool comprises more than approximately 100 differentoligonucleotides.

Further disclosed herein is a microarray having base cleavable sulfonyllinkers. The microarray comprises an array device having a plurality ofknown locations where each location has a plurality of reacted hydroxylgroups. The density of the plurality of known locations is greater thanapproximately 100 per square centimeter. The microarray furthercomprises a plurality of reacted sulfonyl amidite moieties bonded to theplurality of reacted hydroxyl groups to form a plurality of cleavablelinkers attached to the plurality of known locations. The cleavablelinkers have a linker hydroxyl group and a base-labile cleaving site. Aphosphorous-oxygen bond is between phosphorous of the reacted sulfonylamidite moieties and oxygen of the reacted hydroxyl groups.

Further disclosed herein is a microarray having base cleavable sulfonyllinkers. The microarray comprises an array device having a plurality ofknown locations where each location has a plurality of reacted hydroxylgroups. The density of the plurality of known locations is greater thanapproximately 100 per square centimeter. The microarray furthercomprises a plurality of reacted sulfonyl amidite moieties bonded to theplurality of reacted hydroxyl groups to form a plurality of cleavablelinkers attached to the plurality of known locations. The cleavablelinkers have a linker hydroxyl group and a base-labile cleaving site. Aphosphorous-oxygen bond is between phosphorous of the reacted sulfonylamidite moieties and oxygen of the reacted hydroxyl groups. Themicroarray further comprises oligomers bonded to the linker hydroxylgroups.

Further disclosed herein is a process for forming a microarray havingcleavable succinate linkers. The process comprises providing a solidsurface having free hydroxyl groups at known locations. The density ofthe known locations is greater than approximately 100 locations persquare centimeter. The process further comprises bonding a linker moietyto the hydroxyl groups. The linker moiety comprises free amine group anda hydroxyl bonding group. The process further comprises bonding asuccinate-containing moiety having free carboxyl groups to the freeamine groups to form cleavable linkers attached to the known locations.The succinate-containing moieties comprise a sugar having both anucleotide base group and a succinate group bonded to the sugar. Thecleavable linkers have a base-labile cleaving site on the succinategroup and a reactable hydroxyl group on the sugar group.

Further disclosed herein is a microarray having base cleavable succinatelinkers. The microarray comprises a solid surface having known locationsand reactive hydroxyl groups. The known locations have a density greaterthan approximately 100 per square centimeter. The microarray furthercomprises a plurality of reactive amino amidite moieties bonded to thereactive hydroxyl groups on the solid surface. The reactive aminomoieties comprise an amine group and a hydroxyl bonding group. Thehydroxyl bonding group is bonded to the reactive hydroxyl groups at theknown locations. The microarray further comprises a plurality ofreactive succinate moieties bonded to the amine groups. The reactivesuccinate moieties comprise a sugar group bonded to the succinate groupand to a base group bonded. In an alternative embodiment, microarrayfurther comprises oligomers bonded onto the reactable hydroxyl groups.In one or more embodiments, the sugar group is ribose and the base groupis selected from the group consisting of adenine, guanine, cytosine, anduracyl, or the sugar group is deoxyribose and the base group is selectedfrom the group consisting of adenine, guanine, cytosine, and thymine.

Further disclosed herein is a process of forming a microarray havingbase cleavable phosphoramidite linkers. The process comprises providinga microarray having a surface with a plurality of known locations on thesurface. Each location has a plurality of hydroxyl groups, and thedensity of the known locations is greater than approximately 100 persquare centimeter on the surface. The process further comprises bondinga plurality of base cleavable phosphoramidite linkers to the pluralityof hydroxyl groups directly or by using an intermediate chemical moietyattached to the hydroxyl groups to form a plurality of cleavable linkersat the plurality of known locations. The cleavable linkers each have alinker hydroxyl group and a base-labile cleaving site. The linkerhydroxyl group is protected by a protecting group, and the base-labilecleaving site is an ether linkage. Optionally, the process furthercomprises synthesizing oligomers onto the linker hydroxyl groups toprovide a microarray of oligomers. The protecting group is removed fromthe linker hydroxyl groups before synthesizing the oligomers, and theoligomers at the known locations, as between different known locations,are different or the same. Optionally, the process further comprisescleaving at the base-labile cleaving site the oligomers from the surfaceusing a cleaving base to provide a pool of cleaved oligomers.

Further disclosed herein is a pool of oligomers produced according toone or more of the processes disclosed herein, wherein the oligomers areoligonucleotides having a 3′ phosphate, wherein the pool comprises morethan approximately 100 different oligonucleotides. Further disclosedherein is a pool of oligomers produced according to one or more of theprocesses disclosed herein, wherein the oligomers are oligonucleotideshaving a 3′ hydroxyl, wherein the pool comprises more than approximately100 different oligonucleotides.

Further disclosed herein is a microarray having base cleavable linkers.The microarray comprises a microarray having a surface with a pluralityof known locations on the surface, wherein each location has a pluralityof hydroxyl groups, wherein the density of the known locations isgreater than approximately 100 per square centimeter on the surface. Themicroarray further comprises a plurality of base cleavable linkersbonded to the plurality of hydroxyl groups to form a plurality ofcleavable linkers at the plurality of known locations, wherein thecleavable linkers each have a linker hydroxyl group and a base-labilecleaving site.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are a schematic of a cross section of a microarraydevice showing one known location undergoing a sequence of steps for thebonding of a cleavable linker, an oligonucleotide to the linker, andthen removal of the oligonucleotide by cleaving the linker using a base.

FIG. 2 is a schematic of a sulfonyl amidite used to form a cleavablelinker on a micro array.

FIG. 3 is an image of a gel from a gel electrophoresis ofoligonucleotides recovered from three different microarrays having thecleavable sulfonyl linker.

FIG. 4 shows an image of a portion of the microarray after exposure tothe fluorescently labeled oligonucleotide. There are four differentareas, A, B, C, and D, shown in the figure. In areas A, B, and C,oligonucleotides were synthesized with and without a cleavable linker.As can be seen in the figure, the microarray locations having thecleavable linker between the oligonucleotide and the microarray aredark, indicating little or no hybridizable oligonucleotide remainedafter cleaving. In contrast, those locations that did not have thecleavable linker between the oligonucleotide and the microarray arebrighter, which indicates that the oligonucleotide remained on themicroarray. In area D, some electrodes had cleavable linker while othersdid not; however, no oligonucleotides were synthesized so that theentire area appears dark.

FIGS. 5A and 5B are schematics showing the construction of themicroarray of the present invention.

FIG. 6 provides exemplary compounds used to construct the microarray ofthe present invention.

FIG. 7 provides an image of the results from gel electrophoresis of DNAstrands that were amplified by PCR. The image shows recovery of thethree different DNA strands from a microarray after cleaving the strandsfrom a cleavable linker. The DNA strands were synthesized in situ usingelectrochemical synthesis on the cleavable linker attached to themicroarray.

DETAILED DESCRIPTION

Generally, nomenclature for chemical groups as used herein follows therecommendations of “The International Union for Pure and AppliedChemistry”, Principles of Chemical Nomenclature: a Guide to IUPACRecommendations, Leigh, G. J.; Favre, H. A. and Metanomski, W. V.,Blackwell Science, 1998, the disclosure of which is incorporated byreference herein. Formation of substituted structures is limited by atomvalence requirements.

“Oligomer” means a molecule of intermediate relative molecular mass, thestructure of which essentially comprises a small plurality of unitsderived, actually or conceptually, from molecules of lower relativemolecular mass. A molecule is regarded as having an intermediaterelative molecular mass if it has properties which do vary significantlywith the removal of one or a few of the units. If a part or the whole ofthe molecule has an intermediate relative molecular mass and essentiallycomprises a small plurality of units derived, actually or conceptually,from molecules of lower relative molecular mass, it may be described asoligomeric, or by oligomer used adjectivally. Oligomers are typicallycomprised of a monomer.

The term “co-oligomer” means an oligomer derived from more than onespecies of monomer. The term oligomer includes co-oligomers. A singlestranded DNA molecule consisting of any combination of deoxyadenylate(A), deoxyguanylate (G), deoxycytidylate (C), and deoxythymidylate (T)units is an oligomer.

The term “monomer” means a molecule that can undergo polymerizationthereby contributing constitutional units to the essential structure ofa macromolecule such as an oligomer, co-oligomer, polymer, orco-polymer. Examples of monomers for oligonucleotides include A, C, G,T, adenylate, guanylate, cytidylate, and uridylate. Monomers for otheroligomers, including polypeptides, include amino acids, vinyl chloride,and other vinyls.

The term “polymer” means a substance composed of macromolecules, whichis a molecule of high relative molecular mass, the structure of whichessentially comprises the multiple repetition of units derived, actuallyor conceptually, from molecules of low relative molecular mass. In manycases, especially for synthetic polymers, a molecule can be regarded ashaving a high relative molecular mass if the addition or removal of oneor a few of the units has a negligible effect on the molecularproperties. This statement fails in the case of certain macromoleculesfor which the properties may be critically dependent on fine details ofthe molecular structure. If a part or the whole of the molecule has ahigh relative molecular mass and essentially comprises the multiplerepetition of units derived, actually or conceptually, from molecules oflow relative molecular mass, it may be described as eithermacromolecular or polymeric, or by polymer used adjectivally.

The term “copolymer” means a polymer derived from more than one speciesof monomer. Copolymers that are obtained by copolymerization of twomonomer species are sometimes termed biopolymers, those obtained fromthree monomers terpolymers, those obtained from four monomersquaterpolymers, etc. The term polymer includes co-polymers.

The term “polyethylene glycol” (PEG) means an organic chemical having achain consisting of the common repeating ethylene glycol unit[—CH₂—CH₂—O—]_(n). PEG's are typically long chain organic polymers thatare flexible, hydrophilic, enzymatically stable, and biologically inert,but they do not have an ionic charge in water. In general, PEG can bedivided into two categories. First, there is polymeric PEG having amolecular weight ranging from 1000 to greater than 20,000. Second, thereare PEG-like chains having a molecular weight that is less than 1000.Polymeric PEG has been used in bioconjugates, and numerous reviews havedescribed the attachment of this linker moiety to various molecules. PEGhas been used as a linker, where the short PEG-like linkers can beclassified into two types, the homo-[X—(CH₂—CH₂—O)_(n)]—X andheterobifunctional [X—(CH₂—CH₂—O)_(n)]—Y spacers.

The term “PEG derivative” means an ethylene glycol derivative having thecommon repeating unit of PEG. Examples of PEG derivatives include, butare not limited to, diethylene glycol (DEG), tetraethylene glycol (TEG),polyethylene glycol having primary amino groups, di(ethylene glycol)mono allyl ether, di(ethylene glycol) mono tosylate, tri(ethyleneglycol) mono allyl ether, tri(ethylene glycol) mono tosylate,tri(ethylene glycol) mono benzyl ether, tri(ethylene glycol) mono tritylether, tri(ethylene glycol) mono chloro mono methyl ether, tri(ethyleneglycol) mono tosyl mono allyl ether, tri(ethylene glycol) mono allylmono methyl ether, tetra(ethlyne glycol) mono allyl ether,tetra(ethylene glycol) mono methyl ether, tetra(ethylene glycol) monotosyl mono allyl ether, tetra(ethylene glycol) mono tosylate,tetra(ethylene glycol) mono benzyl ether, tetra(ethylene glycol) monotrityl ether, tetra(ethylene glycol) mono 1-hexenyl ether,tetra(ethylene glycol) mono 1-heptenyl ether, tetra(ethylene glycol)mono 1-octenyl ether, tetra(ethylene glycol) mono 1-decenyl ether,tetra(ethylene glycol) mono 1-undecenyl ether, penta(ethylene glycol)mono methyl ether, penta(ethylene glycol) mono allyl mono methyl ether,penta(ethylene glycol) mono tosyl mono methyl ether, penta(ethyleneglycol) mono tosyl mono allyl ether, hexa(ethylene glycol) mono allylether, hexa(ethylene glycol) mono methyl ether, hexa(ethylene glycol)mono benzyl ether, hexa(ethylene glycol) mono trityl ether,hexa(ethylene glycol) mono 1-hexenyl ether, hexa(ethylene glycol) mono1-heptenyl ether, hexa(ethylene glycol) mono 1-octenyl ether,hexa(ethylene glycol) mono 1-decenyl ether, hexa(ethylene glycol) mono1-undecenyl ether, hexa(ethylene glycol) mono 4-benzophenonyl mono1-undecenyl ether, hepta(ethylene glycol) mono allyl ether,hepta(ethylene glycol) mono methyl ether, hepta(ethylene glycol) monotosyl mono methyl ether, hepta(ethylene glycol) monoallyl mono methylether, octa(ethylene glycol) mono allyl ether, octa(ethylene glycol)mono tosylate, octa(ethylene glycol) mono tosyl mono allyl ether,undeca(ethylene glycol) mono methyl ether, undeca(ethylene glycol) monoallyl mono methyl ether, undeca(ethylene glycol) mono tosyl mono methylether, undeca(ethylene glycol) mono allyl ether, octadeca(ethyleneglycol) mono allyl ether, octa(ethylene glycol), deca(ethylene glycol),dodeca(ethylene glycol), tetradeca(ethylene glycol), hexadeca(ethyleneglycol), octadeca(ethylene glycol), benzophenone-4-hexa(ethylene glycol)allyl ether, benzophenone-4-hexa(ethylene glycol) hexenyl ether,benzophenone-4-hexa(ethylene glycol) octenyl ether,benzophenone-4-hexa(ethylene glycol) decenyl ether,benzophenone-4-hexa(ethylene glycol) undecenyl ether,4-fluorobenzophenone-4′-hexa(ethylene glycol) allyl ether,4-fluorobenzophenone-4′-hexa(ethylene glycol) undecenyl ether,4-hydroxybenzophenone-4′-hexa(ethylene glycol) allyl ether,4-hydroxybenzophenone-4′-hexa(ethylene glycol) undecenyl ether,4-hydroxybenzophenone-4′-tetra(ethylene glycol) allyl ether,4-hydroxybenzophenone-4′-tetra(ethylene glycol) undecenyl ether,4-morpholinobenzophenone-4′-hexa(ethylene glycol) allyl ether,4-morpholinobenzophenone-4′-hexa(ethylene glycol) undecenyl ether,4-morpholinobenzophenone-4′-tetra(ethylene glycol) allyl ether, and4-morpholinobenzophenone-4′-tetra(ethylene glycol) undecenyl ether.

The term “polyethylene glycol having primary amino groups” refers topolyethylene glycol having substituted primary amino groups in place ofthe hydroxyl groups. Substitution can be up to 98% in commercialproducts ranging in molecular weight from 5,000 to 20,000 Da.

The term “alkyl” means a straight or branched chain alkyl groupcontaining up to approximately 20 but preferably up to 8 carbon atoms.Examples of alkyl groups include but are not limited to the following:methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl,tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, isohexyl,n-hexyl, n-heptyl, and n-octyl. A substituted alkyl has one or morehydrogen atoms substituted by other groups or a carbon replaced by adivalent, trivalent, or tetravalent group or atom. Although alkyls bydefinition have a single radical, as used herein, alkyl includes groupsthat have more than one radical to meet valence requirements forsubstitution.

The term “alkenyl” means a straight or branched chain alkyl group havingat least one carbon-carbon double bond, and containing up toapproximately 20 but preferably up to 8 carbon atoms. Examples ofalkenyl groups include, but are not limited to, vinyl, 1-propenyl,2-butenyl, 1,3-butadienyl, 2-pentenyl, 2,4-hexadienyl,4-(ethyl)-1,3-hexadienyl, and 2-(methyl)-3-(propyl)-1,3-butadienyl. Asubstituted alkenyl has one or more hydrogen atoms substituted by othergroups or a carbon replaced by a divalent, trivalent, or tetravalentgroup or atom. Although alkenyls by definition have a single radical, asused herein, alkenyl includes groups that have more than one radical tomeet valence requirements for substitution.

The term “alkynyl” means a straight or branched chain alkyl group havinga single radical, having at least one carbon-carbon triple bond, andcontaining up to approximately 20 but preferably up to 8 carbon atoms.Examples of alkynyl groups include, but are not limited to, the ethynyl,1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 4-pentynyl,5-hexynyl, 6-heptynyl, 7-octynyl, 1-methyl-2-butynyl,2-methyl-3-pentynyl, 4-ethyl-2-pentynyl, and 5,5-methyl-1,3-hexynyl. Asubstituted alkynyl has one or more hydrogen atoms substituted by othergroups or a carbon replaced by a divalent, trivalent, or tetravalentgroup or atom. Although alkynyls by definition have a single radical, asused herein, alkynyl includes groups that have more than one radical tomeet valence requirements for substitution.

The term “cycloalkyl” means an alkyl group forming at least one ring,wherein the ring has approximately 3 to 14 carbon atoms. Examples ofcycloalkyl groups include but are not limited to the following:cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. A substitutedcycloalkyl has one or more hydrogen atoms substituted by other groups ora carbon replaced by a divalent, trivalent, or tetravalent group oratom. Although cycloalkyls by definition have a single radical, as usedherein, cycloalkyl includes groups that have more than one radical tomeet valence requirements for substitution.

The term “cycloalkenyl” means an alkenyl group forming at least one ringand having at least one carbon-carbon double bond within the ring,wherein the ring has approximately 3 to 14 carbon atoms. Examples ofcycloalkenyl groups include, but are not limited to, cyclopropenyl,cyclobutenyl, cyclopentenyl, 1,3-cyclopentadienyl, and cyclohexenyl. Asubstituted cycloalkenyl has one or more hydrogens substituted by othergroups or a carbon replaced by a divalent, trivalent, or tetravalentgroup or atom. Although cycloalkenyls by definition have a singleradical, as used herein, cycloalkenyl includes groups that have morethan one radical to meet valence requirements for substitution.

The term “cycloalkynyl” means an alkynyl group forming at least one ringand having at least one carbon-carbon triple bond, wherein the ringcontains up to approximately 14 carbon atoms. A group forming a ringhaving at least one triple bond and having at least one double bond is acycloalkynyl group. An example of a cycloalkynyl group includes, but isnot limited to, cyclooctyne. A substituted cycloalkynyl has one or morehydrogen atoms substituted by other groups. Although cycloalkynyls bydefinition have a single radical, as used herein, cycloalkynyl includesgroups that have more than one radical to meet valence requirements forsubstitution.

The term “aryl” means an aromatic carbon ring group having a singleradical and having approximately 4 to 20 carbon atoms. Examples of arylgroups include, but are not limited to, phenyl, naphthyl, and anthryl. Asubstituted aryl has one or more hydrogen atoms substituted by othergroups. Although aryls by definition have a single radical, as usedherein, aryl includes groups that have more than one radical to meetvalence requirements for substitution. An aryl group can be a part of afused ring structure such as N-hydroxysuccinimide bonded to phenyl(benzene) to form N-hydroxyphthalimide.

The term “hetero” when used in the context of chemical groups, or“heteroatom” means an atom other than carbon or hydrogen. Preferredexamples of heteroatoms include oxygen, nitrogen, phosphorous, sulfur,boron, silicon, and selenium.

The term “heterocyclic ring” means a ring structure having at least onering moiety having at least one heteroatom forming a part of the ring,wherein the heterocyclic ring has approximately 4 to 20 atoms connectedto form the ring structure. An example of a heterocyclic ring having 6atoms is pyridine with a single hereroatom. Additional examples ofheterocyclic ring structures having a single radical include, but arenot limited to, acridine, carbazole, chromene, imidazole, furan, indole,quinoline, and phosphinoline. Examples of heterocyclic ring structuresinclude, but are not limited to, aziridine, 1,3-dithiolane,1,3-diazetidine, and 1,4,2-oxazaphospholidine. Examples of heterocyclicring structures having a single radical include, but are not limited to,fused aromatic and non-aromatic structures: 2H-furo[3,2-b]pyran,5H-pyrido[2,3-d]-o-oxazine, 1H-pyrazolo[4,3-d]oxazole,4H-imidazo[4,5-d]thiazole, selenazolo[5,4-j]benzothiazole, andcyclopenta[b]pyran. Heterocyclic rings can have one or more radicals tomeet valence requirements for substitution.

The term “polycyclic” or “polycyclic group” means a carbon ringstructure having more than one ring, wherein the polycyclic group hasapproximately 4 to 20 carbons forming the ring structure and has asingle radical. Examples of polycyclic groups include, but are notlimited to, bicyclo[1.1.0]butane, bicyclo[5.2.0]nonane, andtricycle[5.3.1.1]dodecane. Polycyclic groups can have one or moreradicals to meet valence requirements for substitution.

The term “halo” or “halogen” means fluorine, chlorine, bromine, oriodine.

The term “heteroatom group” means one heteroatom or more than oneheteroatoms bound together and having two free valences for forming acovalent bridge between two atoms. For example, the oxy radical, —O— canform a bridge between two methyls to form CH₃—O—CH₃ (dimethyl ether) orcan form a bridge between two carbons to form an epoxy such as cis ortrans 2,3-epoxybutane,

As used herein and in contrast to the normal usage, the term heteroatomgroup will be used to mean the replacement of groups in an alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl and not theformation of cyclic bridges, such as an epoxy, unless the term cyclicbridge is used with the term heteroatom group to denote the normalusage.

Examples of heteroatom groups, using the nomenclature for hetero bridges(such as an epoxy bridge), include but are not limited to the following:azimino (—N═N—HN—), azo (—N═N—), biimino (—NH—NH—), epidioxy (—O—O—),epidithio (—S—S—), epithio (—S—), epithioximino (—S—O—NH—), epoxy (—O—),epoxyimino (—O—NH—), epoxynitrilo (—O—N═), epoxythio (—O—S—),epoxythioxy(—O—S—O—), furano (—CH₂O—), imino (—NH—), and nitrilo (—N═).Examples of heteroatom groups using the nomenclature for forming acyclicbridges include but are not limited to the following: epoxy (—O—),epithio (—S—), episeleno (—Se—), epidioxy (—O—O—), epidithio (—S—S—),lambda⁴-sulfano (—SH₂—), epoxythio (—O—S—), epoxythioxy (—O—S—O—),epoxyimino (—O—NH—), epimino (—NH—), diazano (—NH—NH—), diazeno (—N═N—),triaz[1]eno (—N═N—NH—), phosphano (—PH—), stannano (—SnH₂—),epoxymethano (—O—CH₂—), epoxyethano (—O—CH₂—CH₂—), epoxyprop[1]eno

The term “bridge” means a connection between one part of a ringstructure to another part of the ring structure by a hydrocarbon bridge.Examples of bridges include but are not limited to the following:methano, ethano, etheno, propano, butano, 2-buteno, and benzeno.

The term “hetero bridge” means a connection between one part of a ringstructure to another part of the ring structure by one or moreheteroatom groups, or a ring formed by a heterobridge connecting onepart of a linear structure to another part of the linear structure, thusforming a ring.

The term “oxy” means the divalent radical —O—.

The term “oxo” means the divalent radical ═O.

The term “carbonyl” means the group

wherein the carbon has two radicals for bonding.

The term “amide” or “acylamino” means the group

wherein the nitrogen has one single radical for bonding and R ishydrogen or an unsubstituted or substituted alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, orpolycyclic group.

The term “alkoxy” means the group —O—R, wherein the oxygen has a singleradical and R is hydrogen or an unsubstituted or substituted alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,heterocyclic ring, or polycyclic group. Examples of alkoxy groups wherethe R is an alkyl include but are not limited to the following: methoxy,ethoxy, propoxy, butoxy, pentoxy, hexoxy, heptoxy, octoxy,1,1-dimethylethoxy, 1,1-dimethylpropoxy, 1,1-dimethylbutoxy,1,1-dimethylpentoxy, 1-ethyl-1-methylbutoxy, 2,2-dimethylpropoxy,2,2-dimethylbutoxy, 1-methyl-1-ethylpropoxy, 1,1-diethylpropoxy,1,1,2-trimethylpropoxy, 1,1,2-trimethylbutoxy,1,1,2,2-tetramethylpropoxy. Examples of alkoxy groups where the R is analkenyl group include but are not limited to the following: ethenyloxy,1-propenyloxy, 2-propenyloxy, 1-butenyloxy, 2-butenyloxy, 3-butenyloxy,1-methyl-prop-2-enyloxy, 1,1-dimethyl-prop-2-enyloxy,1,1,2-trimethyl-prop-2-enyloxy, and 1,1-dimethyl-but-2-enyloxy,2-ethyl-1,3-dimethyl-but-1-enyloxy. Examples of alkyloxy groups wherethe R is an alkynyl include but are not limited to the following:ethynyloxy, 1-propynyloxy, 2-propynyloxy, 1-butynyloxy, 2-butynyloxy,3-butynyloxy, 1-methyl-prop-2-ynyloxy, 1,1-dimethyl-prop-2-ynyloxy, and1,1-dimethyl-but-2-ynyloxy, 3-ethyl-3-methyl-but-1-ynyloxy. Examples ofalkoxy groups where the R is an aryl group include but are not limitedto the following: phenoxy, 2-naphthyloxy, and 1-anthyloxy.

The term “acyl” means the group

wherein the carbon has a single radical and R is hydrogen or anunsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclicgroup. Examples of acyl groups include but are not limited to thefollowing: acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl,acryloyl, propioloyl, mathacryloyl, crotonoyl, isocrotonoyl, benzoyl,and naphthoyl.

The term “acyloxy” means the group

wherein the oxygen has a single radical and R is hydrogen or anunsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclicgroup. Examples of acyloxy groups include but are not limited to thefollowing: acetoxy, ethylcarbonyloxy, 2-propenylcarbonyloxy,pentylcarbonyloxy, 1-hexynylcarbonyloxy, benzoyloxy,cyclohexylcarbonyloxy, 2-naphthoyloxy, 3-cyclodecenylcarbonyloxy.

The term “oxycarbonyl” means the group

wherein the carbon has a single radical and R is hydrogen or anunsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclicgroup. Examples of oxycarbonyl groups include but are not limited to thefollowing: methoxycarbonyl, ethoxycarbonyl, isopropyloxycarbonyl,phenoxycarbonyl, and cyclohexyloxycarbonyl.

The term “acyloxycarbonyl” means the group

wherein the carbon has a single radical and R is hydrogen or anunsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclicgroup.

The term “alkoxycarbonyloxy” means the group

wherein the oxygen has a single radical and R is hydrogen or anunsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclicgroup.

The term “carboxy” means the group —C(O)OH, wherein the carbon has asingle radical.

The term “imino” or “nitrene” means the group ═N—R, wherein the nitrogenhas two radicals and R is hydrogen or an unsubstituted or substitutedalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,heterocyclic ring, or polycyclic group.

The term “amino” means the group —NH2, where the nitrogen has a singleradical.

The term “secondary amino” means the group —NH—R, wherein the nitrogenhas a single radical and R is hydrogen or an unsubstituted orsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,cycloalkynyl, aryl, heterocyclic ring, or polycyclic group.

The term “tertiary amino” means the group

wherein the nitrogen has a single radical and R1 and R2 areindependently selected from the group consisting of unsubstituted andsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,cycloalkynyl, aryl, heterocyclic ring, and polycyclic group.

The term “hydrazi” means the group —NH—NH—, wherein the nitrogens havesingle radicals bound to the same atom. The term “hydrazo” means thegroup —NH—NH—, wherein the nitrogens have single radicals bound to thedifferent atoms.

The term “hydrazino” means the group NH₂—NH—, wherein the nitrogen has asingle radical.

The term “hydrazono” means the group NH₂—N═, wherein the nitrogen hastwo radicals.

The term “hydroxyimino” means the group HO—N═, wherein the nitrogen hastwo radicals.

The term “alkoxyimino” means the group R—O—N═, wherein the nitrogen hastwo radicals and R is an unsubstituted or substituted alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclicring, or polycyclic group.

The term “azido” means the group N₃—, wherein the nitrogen has oneradical.

The term “azoxy” means the group —N(O)═N—, wherein the nitrogens haveone radical.

The term “alkazoxy” means the group R—N(O)═N—, wherein the nitrogen hasone radical and R is hydrogen or an unsubstituted or substituted alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,heterocyclic ring, or polycyclic group. Azoxybenzene is an examplecompound.

The term “cyano” means the group —CN. The term “isocyano” means thegroup —NC. The term “cyanato” means the group —OCN. The term“isocyanato” means the group —NCO. The term “fulminato” means the group—ONC. The term “thiocyanato” means the group —SCN. The term“isothiocyanato” means the group —NCS. The term “selenocyanato” meansthe group —SeCN. The term “isoselenocyanato” means the group —NCSe.

The term “carboxyamido” or “acylamino” means the group

wherein the nitrogen has a single radical and R is hydrogen or anunsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclicgroup.

The term “acylimino” means the group

wherein the nitrogen has two radicals and R is hydrogen or anunsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclicgroup.

The term “nitroso” means the group O═N—, wherein the nitrogen has asingle radical.

The term “aminooxy” means the group —O—NH₂, wherein the oxygen has asingle radical.

The term “carxoimidioy” means the group

wherein the carbon has a single radical and R is hydrogen or anunsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclicgroup.

The term “hydrazonoyl” means the group

wherein the carbon has a single radical and R is hydrogen or anunsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclicgroup.

The term “hydroximoyl” or “oxime” means the group

wherein the carbon has a single radical and R is hydrogen or anunsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclicgroup.

The term “hydrazino” means the group

wherein the nitrogen has a single radical and R is hydrogen or anunsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclicgroup.

The term “amidino” means the group

wherein the carbon has a single radical.

The term “sulfide” means the group —S—R, wherein the sulfur has a singleradical and R is hydrogen or an unsubstituted or substituted alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,heterocyclic ring, or polycyclic group.

The term “thiol” means the group —S—, wherein the sulfur has tworadicals. Hydrothiol means —SH.

The term “thioacyl” means the group —C(S)—R, wherein the carbon has asingle radical and R is hydrogen or an unsubstituted or substitutedalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,heterocyclic ring, or polycyclic group.

The term “sulfoxide” means the group

wherein the sulfur has a single radical and R is hydrogen or anunsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclicgroup. The term “thiosulfoxide” means the substitution of sulfur foroxygen in sulfoxide; the term includes substitution for an oxygen boundbetween the sulfur and the R group when the first carbon of the R grouphas been substituted by an oxy group and when the sulfoxide is bound toa sulfur atom on another group.

The term “sulfone” means the group

wherein the sulfur has a single radical and R is hydrogen or anunsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclicgroup. The term “thiosulfone” means substitution of sulfur for oxygen inone or two locations in sulfone; the term includes substitution for anoxygen bound between the sulfur and the R group when the first carbon ofthe R group has been substituted by an oxy group and when the sulfone isbound to a sulfur atom on another group.

The term “sulfate” means the group

wherein the oxygen has a single radical and R is hydrogen or anunsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclicgroup. The term “thiosulfate” means substitution of sulfur for oxygen inone, two, three, or four locations in sulfate.

The term “phosphoric acid ester” means the group R¹R²PO₄—, wherein theoxygen has a single radical and R¹ is selected from the group consistingof hydrogen and unsubstituted and substituted alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, andpolycyclic group, and R² is selected from the group consisting ofunsubstituted and substituted alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, and polycyclicgroup.

The term “substituted” or “substitution,” in the context of chemicalspecies, means independently selected from the group consisting of (1)the replacement of a hydrogen on at least one carbon by a monovalentradical, (2) the replacement of two hydrogens on at least one carbon bya divalent radical, (3) the replacement of three hydrogens on at leastone terminal carbon (methyl group) by a trivalent radical, (4) thereplacement of at least one carbon and the associated hydrogens (e.g.,methylene group) by a divalent, trivalent, or tetravalent radical, and(5) combinations thereof. Meeting valence requirements restrictssubstitution. Substitution occurs on alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, andpolycyclic groups, providing substituted alkyl, substituted alkenyl,substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl,substituted cycloalkynyl, substituted aryl group, substitutedheterocyclic ring, and substituted polycyclic groups.

The groups that are substituted on an alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, andpolycyclic groups are independently selected from the group consistingof alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl,aryl, heterocyclic ring, polycyclic group, halo, heteroatom group, oxy,oxo, carbonyl, amide, alkoxy, acyl, acyloxy, oxycarbonyl,acyloxycarbonyl, alkoxycarbonyloxy, carboxy, imino, amino, secondaryamino, tertiary amino, hydrazi, hydrazino, hydrazono, hydroxyimino,azido, azoxy, alkazoxy, cyano, isocyano, cyanato, isocyanato,thiocyanato, fulminato, isothiocyanato, isoselenocyanato, selenocyanato,carboxyamido, acylimino, nitroso, aminooxy, carboximidoyl, hydrazonoyl,oxime, acylhydrazino, amidino, sulfide, thiol, sulfoxide, thiosulfoxide,sulfone, thiosulfone, sulfate, thiosulfate, hydroxyl, formyl,hydroxyperoxy, hydroperoxy, peroxy acid, carbamoyl, trimethyl silyl,nitrilo, nitro, aci-nitro, nitroso, semicarbazono, oxamoyl, pentazolyl,seleno, thiooxi, sulfamoyl, sulfenamoyl, sulfeno, sulfinamoyl, sulfino,sulfinyl, sulfo, sulfoamino, sulfonato, sulfonyl, sulfonyldioxy,hydrothiol, tetrazolyl, thiocarbamoyl, thiocarbazono, thiocarbodiazono,thiocarbonohydrazido, thiocarbonyl, thiocarboxy, thiocyanato,thioformyl, thioacyl, thiosemicarbazido, thiosulfino, thiosulfo,thioureido, thioxo, triazano, triazeno, triazinyl, trithio,trithiosulfo, sulfinimidic acid, sulfonimidic acid, sulfinohydrazonicacid, sulfonohydrazonic acid, sulfinohydroximic acid, sulfonohydroximicacid, and phosphoric acid ester, and combinations thereof.

As an example of a substitution, replacement of one hydrogen atom onethane by a hydroxyl provides ethanol, and replacement of two hydrogensby an oxo on the middle carbon of propane provides acetone (dimethylketone.) As a further example, replacement the middle carbon (themethenyl group) of propane by the oxy radical (—O—) provides dimethylether (CH₃—O—OCH₃.) As a further example, replacement of one hydrogenatom on benzene by a phenyl group provides biphenyl.

As provided above, heteroatom groups can be substituted inside an alkyl,alkenyl, or alkylnyl group for a methylene group (:CH₂) thus forming alinear or branched substituted structure rather than a ring or can besubstituted for a methylene inside of a cycloalkyl, cycloalkenyl, orcycloalkynyl ring thus forming a heterocyclic ring. As a furtherexample, nitrilo (—N═) can be substituted on benzene for one of thecarbons and associated hydrogen to provide pyridine, or and oxy radicalcan be substituted to provide pyran.

The term “unsubstituted” means that no hydrogen or carbon has beenreplaced on an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,cycloalkynyl, or aryl group.

The term “linker” means a molecule having one end attached or capable ofattaching to a solid surface and the other end having a reactive groupthat is attached or capable of attaching to a chemical species ofinterest such as a small molecule, an oligomer, or a polymer. A linkermay already be bound to a solid surface and/or may already have achemical species of interest bound to its reactive group. A linker mayhave a protective group attached to its reactive group, where theprotective group is chemically or electrochemically removable. A linkermay comprise more than one molecule, where the molecules are covalentlyjoined in situ to form the linker having the desired reactive groupprojecting away from a solid surface.

The term “spacer” or “linker moiety” means a molecule having one endattached or capable of attaching to the reactive group of a linker orporous reaction layer and the other end having a reactive group that isattached or capable of attaching to a chemical species of interest suchas a small molecule, an oligomer, or a polymer. A spacer may already bebound to a linker or a porous reaction layer and/or may already have achemical species of interest bound to its reactive group. A spacer mayhave a protective group attached to its reactive group, where theprotective group is chemically or electrochemically removable. A spacermay be formed in situ on a linker or porous reaction layer. A spacer maybe formed and then attached to a linker already attached to a solidsurface or attached to a porous reaction layer on the solid surface. Aspacer may be externally synthesized on a chemical species of interestfollowed by attachment to a linker already attached to a solid surfaceor attached to a porous reaction layer on the solid surface. A chemicalspecies of interest may be attached to a spacer that is attached to alinker where the entire structure is then attached to a solid surface ata reactive sight on the solid surface. The purpose of a spacer is toextend the distance between a molecule of interest and a solid surface.

The term “combination linker and spacer” means a linker having both theproperties of a linker and a spacer. A combination linker and spacer maybe synthesized in situ or synthesized externally and attached to a solidsurface.

The term “coating” means a thin layer of material that is chemicallyand/or physically bound to a solid surface. A coating may be attached toa solid surface by mechanical interlocking as well as by van der Waalsforces (dispersion forces and dipole forces), electron donor-acceptorinteractions, metallic coordination/complexation, covalent bonding, or acombination of the aforementioned. A coating can provide a reactivegroup for direct attachment of a chemical species of interest,attachment of a linker, or attachment of a combination linker andspacer. A coating can be polymerized and/or cross-linked in situ.

The term “reactive” or “reaction” as used in reactive or reactioncoating or reactive or reaction layer means that there is a chemicalspecies or bound group within the layer that is capable of forming acovalent bond for attachment of a linker, spacer, or other chemicalspecies to the layer or coating.

The term “porous” as used in porous reactive layer or coating means thatthere are non-uniformities within the layer or coating to allowmolecular species to diffuse into and through the layer or coating.

The term “adsorption” or “adsorbed” means a chemical attachment by vander Waals forces (dispersion forces and dipole forces), electrondonor-acceptor interactions, or metallic coordination/complexation, or acombination of the aforementioned forces. After adsorption, a speciesmay covalently bind to a surface, depending on the surface, the species,and the environmental conditions.

The term “microarray” refers to, in general, planer surface havingspecific spots that are usually arranged in a column and row format,wherein each spot can be used for some type of chemical or biochemicalanalysis, synthesis, or method. The spots on a microarray are typicallysmaller than 100 micrometers. The term “electrode microarray” refers toa microarray of electrodes, wherein the electrodes are the specificspots on the microarray.

The term “synthesis quality” refers to, in general, the average degreeof similarity between a desired or designed chemical or biochemicalspecies and the species actually synthesized. The term can refer toother issues in a synthesis such as the effect of a layer or coating onthe synthesis quality achieved.

The term “salvation” means a chemical process in which solvent moleculesand molecules or ions of a solute combine to form a compound, whereinthe compound is generally a loosely bound complex held together by vander Waals forces (dispersion forces and dipole forces), acid-baseinteractions (electron donor acceptor interactions), ionic interaction,or metal complex interactions but not covalent bonds. In water, the pHof the water can affect solvation of dissociable species such as acidsand bases. In addition, the concentration of salts as well as the chargeon salts can affect salvation.

The term “agarose” means any commercially available agarose. Agarose isa polysaccharide biopolymer and is usually obtained from seaweed.Agarose has a relatively large number of hydroxyl groups, which providefor high water solubility. Agarose is available commercially in a wideranger of molecular weights and properties.

The term “controlled pore glass” means any commercially availablecontrolled pore glass material suitable for coating purposes. Ingeneral, controlled pore glass (CPG) is an inorganic glass materialhaving a high surface area owing to a large amount of void space.

The term “monosaccharide” means one sugar molecule unlinked to any othersugars. Examples of monosaccharides include allose, altrose, arabinose,deoxyribose, erythrose, fructose (D-Levulose), galactose, glucose,gulose, idose, lyxose, mannose, psicose, ribose, ribulose,sedoheptulose, D-sorbitol, sorbose, sylulose, L-rhamnose(6-Deoxy-L-mannose), tagatose, talose, threose, xylulose, and xylose.

The term “disaccharide” means two sugars linked together to form onemolecule. Examples of disaccharides include amylose, cellobiose(4-β-D-glucopyranosyl-D-glucopyranose), lactose, maltose(4-O-α-D-glucopyranosyl-D-glucose), melibiose(6-O-α-D-Galactopyranosyl-D-glucose), palatinose(6-O-α-D-Glucopyranosyl-D-fructose), sucrose, and trehalose(α-D-Glucopyranosyl-α-D-glucopyranoside).

The term “trisaccharide” means three sugars linked together to form onemolecule. Examples of a trisaccharides include raffinose(6-O-α-D-Galactopyranosyl-D-glucopyranosyl-β-D-fructofuranoside) andmelezitose(O-α-D-glucopyranosyl-(1→3)-β-D-fructofuranosyl-α-D-glucopyranoside).

The term “polysaccharide” means more than three sugars linked togetherto form one molecule, but more accurately means a sugar-based polymer oroligomer. Examples of polysaccharides include inulin, dextran, starches,and cellulose. Dextran is a polymer composed of glucose subunits (mers.)

The term “linker hydroxyl group” means a hydroxyl group on a linkermoiety, wherein the hydroxyl group is initially protected by aprotecting group such as MMT or DMT. After deprotection, the hydroxylgroup becomes reactable. For example, after deprotection, aphosphoramidite may be bonded to the linker hydroxyl group to form asynthetic oligonucleotide.

Disclosed herein is a process for forming a microarray having basecleavable sulfonyl linkers. The process comprises providing an arrayhaving known locations having a plurality of hydroxyl groups. The arraycomprises a surface or a matrix proximate to the surface, wherein thedensity of the known locations is greater than approximately 100 persquare centimeter. The process further comprises bonding one or aplurality of sulfonyl amidite containing reagents to the hydroxyl groupsat the known locations to form a plurality of cleavable linkers bondedto the known locations. The cleavable linkers comprise a hydroxyl moietyand a base-labile cleaving moiety. A phosphorous-oxygen bond is formedbetween phosphorous of the sulfonyl amidite containing reagent andoxygen of the hydroxyl groups.

Further disclosed herein is a process for forming a microarray havingbase cleavable sulfonyl linkers. The process comprises providing anarray device having a plurality of known locations, each having aplurality of hydroxyl groups. The density of the known locations isgreater than approximately 100 per square centimeter. The processfurther comprises bonding a plurality of sulfonyl amidite moieties tothe hydroxyl groups to form a plurality of cleavable linkers attached tothe array device at each known location. The cleavable linkers comprisea linker hydroxyl moiety and a base-labile cleaving moiety. Aphosphorous-oxygen bond is formed between phosphorous of the sulfonylamidite moieties and oxygen of the hydroxyl groups. The process furthercomprises synthesizing a plurality of oligomers onto the linker hydroxylmoieties.

Further disclosed herein is a process for forming a microarray havingbase cleavable sulfonyl linkers. The process comprises providing anarray device having a plurality of known locations, each having aplurality of hydroxyl groups. The density of the plurality of knownlocations is greater than approximately 100 per square centimeter. Theprocess further comprises bonding a plurality of sulfonyl amiditemoieties to the hydroxyl groups to form a plurality of cleavable linkersbonded to the known locations. The cleavable linkers comprise a linkerhydroxyl moiety and a base-labile cleaving moiety. A phosphorous-oxygenbond is formed between phosphorous of the sulfonyl amidite moieties andoxygen of the hydroxyl groups. The process further comprisessynthesizing a plurality of oligomers covalently bound to the linkerhydroxyl moiety. The process further comprises cleaving the oligomersfrom the known locations at the base-labile cleaving moiety using acleaving base. The oligomers are recoverable. The oligomers comprisingDNA and RNA have a 3′ phosphate after cleaving from the solid surface.

In one or more embodiments, the cleaving base is selected from the groupconsisting of ammonium hydroxide, electrochemically generated base,sodium hydroxide, potassium hydroxide, methylamine, and ethylamine andcombinations thereof.

Further disclosed herein is a process for forming a pool of oligomersproduced by providing an array having known locations having a pluralityof hydroxyl groups. The array comprises a surface or a matrix proximateto the surface. The density of the known locations is greater thanapproximately 100 per square centimeter. The process further comprisesbonding a plurality of sulfonyl amidite moieties to the hydroxyl groupsto form a plurality of cleavable linkers bonded to the known locations.The cleavable linkers comprise a linker hydroxyl moiety and abase-labile cleaving moiety. A phosphorous-oxygen bond is formed betweenphosphorous of the sulfonyl amidite moieties and oxygen of the hydroxylgroups. The process further comprises synthesizing a plurality ofoligomers covalently bound to the linker hydroxyl moiety. The processfurther comprises cleaving the oligomers from the known locations at thebase-labile cleaving moiety using a cleaving base. The oligomerscomprise DNA and RNA and have a 3′ phosphate after cleaving from thesolid surface. The oligomers are oligonucleotides having a 3′ phosphate.The pool comprises more than approximately 100 differentoligonucleotides.

Further disclosed herein is a microarray having base cleavable sulfonyllinkers. The microarray comprises an array device having a plurality ofknown locations where each location has a plurality of reacted hydroxylgroups. The density of the plurality of known locations is greater thanapproximately 100 per square centimeter. The microarray furthercomprises a plurality of reacted sulfonyl amidite moieties bonded to theplurality of reacted hydroxyl groups to form a plurality of cleavablelinkers attached to the plurality of known locations. The cleavablelinkers have a linker hydroxyl group and a base-labile cleaving site. Aphosphorous-oxygen bond is between phosphorous of the reacted sulfonylamidite moieties and oxygen of the reacted hydroxyl groups.

Further disclosed herein is a microarray having base cleavable sulfonyllinkers. The microarray comprises an array device having a plurality ofknown locations where each location has a plurality of reacted hydroxylgroups. The density of the plurality of known locations is greater thanapproximately 100 per square centimeter. The microarray furthercomprises a plurality of reacted sulfonyl amidite moieties bonded to theplurality of reacted hydroxyl groups to form a plurality of cleavablelinkers attached to the plurality of known locations. The cleavablelinkers have a linker hydroxyl group and a base-labile cleaving site. Aphosphorous-oxygen bond is between phosphorous of the reacted sulfonylamidite moieties and oxygen of the reacted hydroxyl groups. Themicroarray further comprises oligomers bonded to the linker hydroxylgroups.

Further disclosed herein is a process for forming a microarray havingcleavable succinate linkers. The process comprises providing a solidsurface having free hydroxyl groups at known locations. The density ofthe known locations is greater than approximately 100 locations persquare centimeter. The process further comprises bonding a linker moietyto the hydroxyl groups. The linker moiety comprises free amine group anda hydroxyl bonding group. The process further comprises bonding asuccinate-containing moiety having free carboxyl groups to the freeamine groups to form cleavable linkers attached to the known locations.The succinate-containing moieties comprise a sugar having both anucleotide base group and a succinate group bonded to the sugar. Thecleavable linkers have a base-labile cleaving site on the succinategroup and a reactable hydroxyl group on the sugar group. In one or moreembodiments, the sugar moiety has one or a plurality of free hydroxylgroups. In another embodiment, the process further comprisessynthesizing oligomers, such as oligonucleotides, attached to freehydroxyl groups of the sugar moiety. In another embodiment, the processfurther comprises cleaving oligomers at the base-labile cleaving sitefrom the known location using a cleaving base, whereby the oligomers arerecoverable.

Further disclosed herein is a microarray having base cleavable succinatelinkers. The microarray comprises a solid surface having known locationsand reactive hydroxyl groups. The known locations have a density greaterthan approximately 100 per square centimeter. The microarray furthercomprises a plurality of reactive amino amidite moieties bonded to thereactive hydroxyl groups on the solid surface. The reactive aminomoieties comprise an amine group and a hydroxyl bonding group. Thehydroxyl bonding group is bonded to the reactive hydroxyl groups at theknown locations. The microarray further comprises a plurality ofreactive succinate moieties bonded to the amine groups. The reactivesuccinate moieties comprise a sugar group bonded to the succinate groupand to a base group bonded. In an alternative embodiment, microarrayfurther comprises oligomers bonded onto the reactable hydroxyl groups.In one or more embodiments, the sugar group is ribose and the base groupis selected from the group consisting of adenine, guanine, cytosine, anduracyl, or the sugar group is deoxyribose and the base group is selectedfrom the group consisting of adenine, guanine, cytosine, and thymine.

Further disclosed herein is a process of forming a microarray havingbase cleavable phosphoramidite linkers. The process comprises providinga microarray having a surface with a plurality of known locations on thesurface. Each location has a plurality of hydroxyl groups, and thedensity of the known locations is greater than approximately 100 persquare centimeter on the surface. The process further comprises bondinga plurality of base cleavable phosphoramidite linkers to the pluralityof hydroxyl groups directly or by using an intermediate chemical moietyattached to the hydroxyl groups to form a plurality of cleavable linkersat the plurality of known locations. The cleavable linkers each have alinker hydroxyl group and a base-labile cleaving site. The linkerhydroxyl group is protected by a protecting group, and the base-labilecleaving site is an ether linkage.

Further disclosed herein is a process of forming a microarray havingbase cleavable phosphoramidite linkers. The process comprises providinga microarray having a surface with a plurality of known locations on thesurface. Each location has a plurality of hydroxyl groups, and thedensity of the known locations is greater than approximately 100 persquare centimeter on the surface. The process further comprises bondinga plurality of base cleavable phosphoramidite linkers to the pluralityof hydroxyl groups directly or by using an intermediate chemical moietyattached to the hydroxyl groups to form a plurality of cleavable linkersat the plurality of known locations. The cleavable linkers each have alinker hydroxyl group and a base-labile cleaving site. The linkerhydroxyl group is protected by a protecting group, and the base-labilecleaving site is an ether linkage. The process further comprisessynthesizing oligomers onto the linker hydroxyl groups to provide amicroarray of oligomers. The protecting group is removed from the linkerhydroxyl groups before synthesizing the oligomers, and the oligomers atthe known locations, as between different known locations, are differentor the same.

Further disclosed herein is a process of forming a microarray havingbase cleavable phosphoramidite linkers. The process comprises providinga microarray having a surface with a plurality of known locations on thesurface. Each location has a plurality of hydroxyl groups, and thedensity of the known locations is greater than approximately 100 persquare centimeter on the surface. The process further comprises bondinga plurality of base cleavable phosphoramidite linkers to the pluralityof hydroxyl groups directly or by using an intermediate chemical moietyattached to the hydroxyl groups to form a plurality of cleavable linkersat the plurality of known locations. The cleavable linkers each have alinker hydroxyl group and a base-labile cleaving site. The linkerhydroxyl group is protected by a protecting group, and the base-labilecleaving site is an ether linkage. The process further comprisessynthesizing oligomers onto the linker hydroxyl groups to provide amicroarray of oligomers. The protecting group is removed from the linkerhydroxyl groups before synthesizing the oligomers, and the oligomers atthe known locations, as between different known locations, are differentor the same. The process further comprises cleaving at the base-labilecleaving site the oligomers from the surface using a cleaving base toprovide a pool of cleaved oligomers.

Further disclosed herein is a pool of oligomers produced according toone or more of the processes disclosed herein, wherein the oligomers areoligonucleotides having a 3′ phosphate, wherein the pool comprises morethan approximately 100 different oligonucleotides. Further disclosedherein is a pool of oligomers produced according to one or more of theprocesses disclosed herein, wherein the oligomers are oligonucleotideshaving a 3′ hydroxyl, wherein the pool comprises more than approximately100 different oligonucleotides.

In one or more embodiments, the oligomers are selected from the groupconsisting of DNA, RNA, and polypeptides, and combinations thereof. Inone or more embodiments, the cleaving base is selected from the groupconsisting of chemical base, ammonium hydroxide, electrochemicallygenerated base, sodium hydroxide, potassium hydroxide, methylamine, andethylamine and combinations thereof. In one or more embodiments, theoligomers are synthesized in situ using electrochemical synthesis. Inone or more embodiments, the oligomers are synthesized in situ by amethod selected from the group consisting of (i) printing reagents viaink jet or other printing technology and using regular phosphoramiditechemistry, (ii) maskless photo-generated acid controlled synthesis andusing regular phosphoramidite chemistry, (iii) mask-directed parallelsynthesis using photo-cleavage of photolabile protecting groups, and(iv) maskless parallel synthesis using photo-cleavage of photolabileprotecting groups and digital photolithography.

Further disclosed herein is a microarray having base cleavable linkers.The microarray comprises a microarray having a surface with a pluralityof known locations on the surface, wherein each location has a pluralityof hydroxyl groups, wherein the density of the known locations isgreater than approximately 100 per square centimeter on the surface. Themicroarray further comprises a plurality of base cleavable linkersbonded to the plurality of hydroxyl groups to form a plurality ofcleavable linkers at the plurality of known locations, wherein thecleavable linkers each have a linker hydroxyl group and a base-labilecleaving site.

In one or more embodiments, the surface has electrodes and each of theknown locations are associated with one of the electrodes, wherein theelectrodes are electronically addressable. In one or more embodiments,the known locations are on the same surface as the electrodes, on anopposing surface to the electrodes, or on an overlayer over theelectrodes.

In one or more embodiments, the array comprises electrodes and each ofthe known locations has an electrode, wherein the electrodes areelectronically addressable. In one or more embodiments, the knownlocations are on the same surface as the electrodes, on an opposingsurface to the electrodes, or on an overlayer over the electrodes. Anexample of an electrode microarray is a CombiMatrix CustomArray™ 12K,which has over 12,000 electrodes and an electrode density ofapproximately 17,778 electrodes per square centimeter.

In one or more embodiments, the oligomers are selected from the groupconsisting of DNA, RNA, and polypeptide, and combinations thereof.

In one or more embodiments, the oligomers are synthesized in situ usingelectrochemical synthesis. In one or more embodiments, the oligomers aresynthesized in situ by a method selected from the group consisting of(i) printing reagents via ink jet or other printing technology and usingregular phosphoramidite chemistry, (ii) maskless photo-generated acidcontrolled synthesis and using regular phosphoramidite chemistry, (iii)mask-directed parallel synthesis using photo-cleavage of photolabileprotecting groups, and (iv) maskless parallel synthesis usingphoto-cleavage of photolabile protecting groups and digitalphotolithography.

In one or more embodiments, the array is glass having a silane linkingagent having organic hydroxyl groups, wherein the organic hydroxylgroups are the hydroxyl groups of the known locations. Preferably, thesilane linking agent is a chemical selected from the group consisting ofhydroxymethyltriethoxysilane, N-(3-triethoxysilylpropyl)gluconamide,N-(3-triethoxysilylpropyl)-4-hydroxybutyramide,1-trimethoxysilyl-3-propanol, 1-trimethoxysilyl-2,3-propanediol,1-triethoxysilyl-3-propanol, 1-triethoxysilyl-2,3-propanediol,1-trimethoxysilyl-2-ethanol, triethoxysilyl-2-ethanol,trimethoxysilyl-11-undecanol, and triethoxysilyl-11-undecanol andcombinations thereof.

In one or more embodiments, the sulfonyl amidite moiety is2-[2-(4,4′-dimethoxytrityloxy)ethylsulfonyl)ethyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite.

In one or more embodiments, spacers having reactive hydroxyl groups arebound to the hydroxyl moieties, wherein the sulfonyl amidite moietiesare bound to the reactive hydroxyl groups of the spacers. Preferably,the spacer is selected from the group consisting of DNA, RNA,polyethylene glycol, and polypeptides, and combinations thereof.Preferably, the spacer is approximately 1 to 35 mers.

In one or more embodiments, a porous reaction layer attached to theknown locations provides the hydroxyl groups, wherein the porousreaction layer comprises a chemical species or mixture of chemicalspecie, wherein the chemical species is selected from the groupconsisting of monosaccharides, disaccharides, trisaccharides,polyethylene glycol, polyethylene glycol derivative,N-hydroxysuccinimide, formula I, formula II, formula III, formula IV,formula V, formula VI, formula VII, and combinations thereof, whereinformula I is

formula II is

formula III is HOR⁴(OR⁵)_(m)R⁹ formula IV is

formula V is

formula VI is

and formula VII is

wherein in each formula m is an integer from 1 to 4; R¹, R²; R⁷, and R⁸are independently selected from the group consisting of hydrogen, andsubstituted and unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, and polycyclicgroup, and halo, amide, alkoxy, acyl, acyloxy, oxycarbonyl,acyloxycarbonyl, alkoxycarbonyloxy, carboxy, amino, secondary amino,tertiary amino, hydrazino, azido, alkazoxy, cyano, isocyano, cyanato,isocyanato, thiocyanato, fulminato, isothiocyanato, isoselenocyanato,selenocyanato, carboxyamido, acylimino, nitroso, aminooxy,carboximidoyl, hydrazonoyl, oxime, acylhydrazino, amidino, sulfide,sulfoxide, thiosulfoxide, sulfone, thiosulfone, sulfate, thiosulfate,hydroxyl, formyl, hydroxyperoxy, hydroperoxy, peroxy acid, carbamoyl,trimethyl silyl, nitro, nitroso, oxamoyl, pentazolyl, sulfamoyl,sulfenamoyl, sulfeno, sulfinamoyl, sulfino, sulfo, sulfoamino,hydrothiol, tetrazolyl, thiocarbamoyl, thiocarbazono, thiocarbodiazono,thiocarbonohydrazido, thiocarboxy, thioformyl, thioacyl, thiocyanato,thiosemicarbazido, thiosulfino, thiosulfo, thioureido, triazano,triazeno, triazinyl, trithiosulfo, sulfinimidic acid, sulfonimidic acid,sulfinohydrazonic acid, sulfonohydrazonic acid, sulfinohydroximic acid,sulfonohydroximic acid, and phosphoric acid ester; R³ is selected fromthe group consisting of heteroatom group, carbonyl, and substituted andunsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,cycloalkynyl, aryl, heterocyclic ring, and polycyclic group; R⁴ and R⁵are independently selected from the group consisting of methylene,ethylene, propylene, butylene, pentylene, and hexylene; R⁶ forming aring structure with two carbons of succinimide and is selected from thegroup consisting of substituted and unsubstituted alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclicring, and polycyclic group; and R⁹ is selected from the group consistingof amino and hydroxyl.

In one or more embodiments, the monosaccharide is selected from thegroup consisting of allose, altrose, arabinose, deoxyribose, erythrose,fructose, galactose, glucose, gulose, idose, lyxose, mannose, psicose,L-rhamnose, ribose, ribulose, sedoheptulose, D-sorbitol, sorbose,sylulose, tagatose, talose, threose, xylulose, and xylose. In one ormore embodiments, the disaccharide is selected from the group consistingof amylose, cellobiose, lactose, maltose, melibiose, palatinose,sucrose, and trehalose. In one or more embodiments, the trisaccharide isselected from the group consisting of raffinose and melezitose.

In one or more embodiments, the polyethylene glycol derivative isselected from the group consisting of diethylene glycol, tetraethyleneglycol, polyethylene glycol having primary amino groups,2-(2-aminoethoxy) ethanol, ethanol amine, di(ethylene glycol) mono allylether, di(ethylene glycol) mono tosylate, tri(ethylene glycol) monoallyl ether, tri(ethylene glycol) mono tosylate, tri(ethylene glycol)mono benzyl ether, tri(ethylene glycol) mono trityl ether, tri(ethyleneglycol) mono chloro mono methyl ether, tri(ethylene glycol) mono tosylmono allyl ether, tri(ethylene glycol) mono allyl mono methyl ether,tetra(ethlyne glycol) mono allyl ether, tetra(ethylene glycol) monomethyl ether, tetra(ethylene glycol) mono tosyl mono allyl ether,tetra(ethylene glycol) mono tosylate, tetra(ethylene glycol) mono benzylether, tetra(ethylene glycol) mono trityl ether, tetra(ethylene glycol)mono 1-hexenyl ether, tetra(ethylene glycol) mono 1-heptenyl ether,tetra(ethylene glycol) mono 1-octenyl ether, tetra(ethylene glycol) mono1-decenyl ether, tetra(ethylene glycol) mono 1-undecenyl ether,penta(ethylene glycol) mono methyl ether, penta(ethylene glycol) monoallyl mono methyl ether, penta(ethylene glycol) mono tosyl mono methylether, penta(ethylene glycol) mono tosyl mono allyl ether, hexa(ethyleneglycol) mono allyl ether, hexa(ethylene glycol) mono methyl ether,hexa(ethylene glycol) mono benzyl ether, hexa(ethylene glycol) monotrityl ether, hexa(ethylene glycol) mono 1-hexenyl ether, hexa(ethyleneglycol) mono 1-heptenyl ether, hexa(ethylene glycol) mono 1-octenylether, hexa(ethylene glycol) mono 1-decenyl ether, hexa(ethylene glycol)mono 1-undecenyl ether, hexa(ethylene glycol) mono 4-benzophenonyl mono1-undecenyl ether, hepta(ethylene glycol) mono allyl ether,hepta(ethylene glycol) mono methyl ether, hepta(ethylene glycol) monotosyl mono methyl ether, hepta(ethylene glycol) monoallyl mono methylether, octa(ethylene glycol) mono allyl ether, octa(ethylene glycol)mono tosylate, octa(ethylene glycol) mono tosyl mono allyl ether,undeca(ethylene glycol) mono methyl ether, undeca(ethylene glycol) monoallyl mono methyl ether, undeca(ethylene glycol) mono tosyl mono methylether, undeca(ethylene glycol) mono allyl ether, octadeca(ethyleneglycol) mono allyl ether, octa(ethylene glycol), deca(ethylene glycol),dodeca(ethylene glycol), tetradeca(ethylene glycol), hexadeca(ethyleneglycol), octadeca(ethylene glycol), benzophenone-4-hexa(ethylene glycol)allyl ether, benzophenone-4-hexa(ethylene glycol) hexenyl ether,benzophenone-4-hexa(ethylene glycol) octenyl ether,benzophenone-4-hexa(ethylene glycol) decenyl ether,benzophenone-4-hexa(ethylene glycol) undecenyl ether,4-fluorobenzophenone-4′-hexa(ethylene glycol) allyl ether,4-fluorobenzophenone-4′-hexa(ethylene glycol) undecenyl ether,4-hydroxybenzophenone-4-hexa(ethylene glycol) allyl ether,4-hydroxybenzophenone-4-hexa(ethylene glycol) undecenyl ether,4-hydroxybenzophenone-4′-tetra(ethylene glycol) allyl ether,4-hydroxybenzophenone-4′-tetra(ethylene glycol) undecenyl ether,4-morpholinobenzophenone-4′-hexa(ethylene glycol) allyl ether,4-morpholinobenzophenone-4′-hexa(ethylene glycol) undecenyl ether,4-morpholinobenzophenone-4′-tetra(ethylene glycol) allyl ether, and4-morpholinobenzophenone-4′-tetra(ethylene glycol) undecenyl ether.Preferably, the polyethylene glycol has a molecular weight ofapproximately 1,000 to 20,000.

In one or more embodiments, the sugar group is ribose and the nucleotidebase group is selected from the group consisting of adenine, guanine,cytosine, and uracyl, or the sugar group is deoxyribose and the basegroup is selected from the group consisting of adenine, guanine,cytosine, and thymine.

In one or more embodiments, the cleaving base is selected from the groupconsisting of ammonium hydroxide, electrochemically generated base,sodium hydroxide, potassium hydroxide, methylamine, and ethylamine andcombinations thereof, whereby the oligomers comprising DNA and RNA havea 3′ hydroxyl after cleaving from the solid surface.

In one or more embodiments, the amino moiety is selected from the groupconsisting of aminopropyltrimethoxysilane, aminopropyltriethoxysilane,aminopropylmethyldiethoxysilane, aminopropylmethyldiethoxysilanehydrozylate, m-aminophenyltrimethoxysilane,phenylaminopropyltrimethoxysilane,1,1,2,4-tetramethyl-1-sila-2-azacyclopentane,aminoethylaminopropyltrimethoxysilane,aminoethylaminopropyltrimethoxysilane,aminoethylaminopropyltriethoxysilane,aminoethylaminopropylmethyldimethoxysilane,aminoethylaminopropyltrimethoxysilane hydrolyzate,aminoethylaminoisobutylmethyldimethoxysilane,aminoethylaminoisobutylmethyldimethoxysilane,aminoethylaminoisobutylmethyldimethoxysilane hydrolyzate,trimethoxysilylpropyldiethylenetriamine,vinylbenzylethylenediaminepropyltrimethoxysilane monohydrochloride,vinylbenzylethylenediaminepropyltrimethoxysilane,benzylethylenediaminepropyltrimethoxysilane monohydrochloride,benzylethylenediaminepropyltrimethoxysilane, andallylethylenediaminepropyltrimethoxysilane monohydrochloride, andcombinations thereof.

In one or more embodiments, the amino moieties are an amino amiditemoiety selected from the group consisting of3-(trifluoroacetylamino)propyl-(2-cyanoethyl)-N,N-diisopropyl)-phosphoramidite,2-[2-(4-monomethoxytrityl)aminoethoxy]ethyl-(2-cyanoethyl)-N,N-diisopropyl)-phosphoramidite,6-(4-monomethoxytritylamino)hexyl-(2-cyanoethyl)-N,N-diisopropyl)-phosphoramidite,12-(4-monomethoxytritylamino)dodecyl-(2-cyanoethyl)-N,N-diisopropyl)-phosphoramidite,and6-(trifluoroacetylamino)hexyl-(2-cyanoethyl)-N,N-diisopropyl)-phosphoramidite,and combinations thereof.

In one or more embodiments, the succinate moieties are selected from asalt of a chemical selected from the group consisting of5′dimethoxytrityl-N-benzoyl-2′-deoxycytidine-3′-O-succinate,5′dimethyoxytrityl-N-isobutyryl-2′-deoxyguanosine-3′-O-succinate,5′-dimethoxytrityl-thymidine-3′-O-succinate, and5′-dimethoxytrityl-N-benzoyl-2′-deoxyadenosine-3′-O-succinate, andcombinations thereof. Preferably the salt is a pyridium salt of thesuccinate moieties.

FIGS. 1A and 1B provide a sequence of drawings that show a process ofmaking the microarrays recited In one or more embodiments; the figuresare not drawn to scale. The figures show a cross-section of only one ofthe plurality of known locations, preferably located on a solid surfaceof the microarray. Preferably, the density of the plurality of knownlocations is greater than 100 per square centimeter and can beapproximately 1,000 to approximately 1,000,000 locations per squarecentimeter or even higher. The first step in FIG. 1A shows the hydroxylgroups before reaction. The hydroxyl groups are preferably accessiblefor chemical reactions thereto. The second step of FIG. 1A shows thesulfonyl amidite moieties attached to the hydroxyl groups through aphosphorous-oxygen bond between the phosphorous of the sulfonyl amiditemoieties and the oxygen of the hydroxyl groups. A sulfonyl amiditemoiety is shown in FIG. 2. To attach the sulfonyl amidite moiety, amixture of activator and the amidite is made and applied to themicroarray. Preferably, the activator is tetrazole at a concentration ofabout 0.45 molar before mixing. More preferably, the activator is5-ethylthio-1H-tetrazole at a concentration of about 0.25 molar beforemixing. Preferably, the activator is in acetonitrile. Preferably, theconcentration of the amidite is 100 millimolar before mixing.Preferably, the mixture is a one to one mixture by volume. Preferably,the reaction of the amidite proceeds for about 1 to 30 minutes, and morepreferably the reaction proceeds for about 5 minutes. After reaction,the hydroxyl groups are referred to as reacted hydroxyl groups. Thephosphorous is oxidized from phosphorous III to V according to standardphosphoramidite synthesis. Preferably, the oxidation is performed usingOx-T solution and the reaction proceeds for about 10 to 60 seconds, andmore preferably the reaction proceeds for about 30 seconds. The hydroxylgroups that are not reacted are capped. The protecting group on theoxygen of the sulfonyl amidite moieties is removed using acidic reagent.Preferably, acidic reagent is generated electrochemically while beingconfined by scavenging agents or buffers, natural diffusion, and theporous reaction layer, which partially physically limits diffusion. Theprotecting group is preferably dimethoxytrityl (DMT) although,generally, any acid-labile protecting group will work such as thosedisclosed in Montgomery I, II, or III. The resulting structure formscleavable linkers attached to the microarray at known locations. Thecleaving point is shown in the last step of FIG. 1A. FIG. 1B shows anoligonucleotide cleaved from the microarray and having a three primephosphate.

FIG. 1A shows the attachment of the oligomers after synthesis onto thedeprotected hydroxyl of the sulfonyl amidite moieties. Preferably, theoligomers are selected from the group consisting of DNA, RNA, andpolypeptide, and combinations thereof. FIGS. 1A and 1B show theoligomers as oligonucleotides. More preferably, the oligomers are DNA.Preferably, the oligomers are synthesized in situ using electrochemicalsynthesis. Electrochemical synthesis of DNA or RNA uses standardphosphoramidite synthesis and electrochemical deblocking, which iselectrochemical generation of acid for deprotection of each unit of aDNA or RNA strand. Electrochemical deblocking involves turning on anelectrode to generate acidic conditions at the electrode sufficient toremove the protecting group only at that electrode. The acidic reagentmay be confined as disclosed previously for removing DMT on a sulfonylamidite and as disclosed in the Montgomery patents. Removal of theprotecting group allows addition of the next unit (mer). Optionally, theoligomers are synthesized in situ by a method selected from the groupconsisting of (i) printing reagents via ink jet or other printingtechnology and using regular phosphoramidite chemistry, (ii) masklessphoto-generated acid controlled synthesis and using regularphosphoramidite chemistry, (iii) mask-directed parallel synthesis usingphoto-cleavage of photolabile protecting groups, and (iv) masklessparallel synthesis using photo-cleavage of photolabile protecting groupsand digital photolithography.

Preferably, the cleaving base is selected from the group consisting ofammonium hydroxide, electrochemically generated base, sodium hydroxide,potassium hydroxide, methylamine, and ethylamine and combinationsthereof. More preferably, the cleaving base is concentrated ammoniumhydroxide, the reaction temperature is about 65 degrees Celsius, and thereaction time is about four to six hours. During exposure to thecleaving base, cleaving occurs as well as deprotection ofoligonucleotides synthesized on the cleavable linker. To recoveroligonucleotides cleaved from a microarray, the microarray is preferablyplace on ice for about 10 minutes, and if ammonium hydroxide is used, avacuum evaporator is used to remove the ammonium hydroxide from theoligonucleotides. The oligonucleotides may be re-suspended into solutionand cleaned to remove impurities.

Preferably, each of the known locations is associated with an electrodeto form an electrode array, wherein the electrodes are electronicallyaddressable. An example of an electrode microarray is a CombiMatrixCustomArray™ 12 k, which has over 12,000 electrodes and an electrodedensity of approximately 17,778 electrodes per square centimeter.Preferably, the known locations are on the same surface as theelectrodes, on an opposing surface to the electrodes, or on an overlayerover the electrodes.

Optionally, the array comprises a surface that is glass without a silanelinking agent or with a silane linking agent. Preferably, the silanelinking agent has organic hydroxyl groups that are the hydroxyl groupsof the known locations. Preferably, the silane linking agent is achemical selected from the group consisting ofhydroxymethyltriethoxysilane, N-(3-triethoxysilylpropyl)gluconamide,N-(3-triethoxysilylpropyl)-4-hydroxybutyramide,1-trimethoxysilyl-3-propanol, 1-trimethoxysilyl-2,3-propanediol,1-triethoxysilyl-3-propanol, 1-triethoxysilyl-2,3-propanediol,1-trimethoxysilyl-2-ethanol, triethoxysilyl-2-ethanol,trimethoxysilyl-11-undecanol, and triethoxysilyl-11-undecanol andcombinations thereof.

Preferably, the sulfonyl amidite moiety is2-[2-(4,4′-dimethoxytrityloxy)ethylsulfonyl)ethyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite. Optionally,spacers having reactive hydroxyl groups are bound to the hydroxyl groupsof the known locations, wherein the sulfonyl amidite moities are boundto the reactive hydroxyl groups of the spacers. Preferably, the spaceris selected from the group consisting of DNA, RNA, polyethylene glycol,and polypeptides, and combinations thereof. Preferably, the spacer isapproximately 1 to 35 mers. More preferably, the spacer is a 10-T,although A, C, G, or U may be used in the spacer. The T-spacer isconvenient because of a lack of a protecting group on the base. Anoligonucleotide space may be synthesized using electrochemical synthesisor one of the other methods suitable for oligonucleotide synthesis on amicroarray. Final deprotection of an oligonucleotide linker may beaccomplished by using electrochemical generation of acid or by exposureto acidic solution such as Deblock-T solution, which is 3%trichloroacetic acid in dichloromethane.

FIGS. 5A and 5B provide a schematic of the construction of a microarrayfor one or more of the embodiments. The microarray has a solid surfacewith known locations that have hydroxyl groups. The hydroxyl groups areshown in FIG. 5A in the first step as not reacted; however, the secondstep shows the hydroxyl groups reacted. The density of the knownlocations is greater than approximately 100 locations per squarecentimeter. Density of the known locations can be approximately 1,000 to1,000,000 locations per square centimeter. Only one known location withone hydroxyl is shown. Amino moieties are attached to the hydroxylgroups. The attachment is through a phosphorous-oxygen bond between thephosphorous of amino amidite moieties and the oxygen of the hydroxylgroups as shown in the second step of FIG. 5A. Generally, the hydroxylgroups are referred to as reacted hydroxyl groups after attachment ofthe amino moieties. The amino moieties have an amine group and ahydroxyl reactive group. The hydroxyl reactive group bonds to thehydroxyl groups at the known locations.

The succinate moieties are attached to the amino moieties through amidebonds as shown in the last step in FIG. 5A. Prior to attachment of thesuccinate, the microarray is capped to cap unreacted hydroxyl groupsfollowed by deprotection to remove the protecting group on the amine.The protecting group is preferably monomethoxytrityl (MMT) although,generally, any acid-labile protecting group will work such as thosedisclosed in Montgomery I, II, or III, including dimethoxytrityl (DMT).The resulting structure forms cleavable linkers attached to themicroarray. The cleaving point is shown in FIG. 5B. Oligomers areattached to the cleavable linkers as shown in FIG. 5B. If the oligomersare DNA or RNA and cleaved from the microarray, the resultingoligonucleotide has a 3′ hydroxyl. FIG. 5B provides an example structureon a microarray. The succinate moieties have a succinate group bonded toa sugar group and a base bonded to the sugar group.

The amine group on the amino amidite moiety is protected by a protectinggroup. Generally, amino amidite moieties bonded to the surface arereferred to as reacted amino amidite moieties. Such protection groupsmust be removed before a succinate moiety can be reacted to form anamide linkage between the amino amidite and the succinate moiety. Theprotecting groups are removed on an electrode microarray by thegeneration of acidic protons at the locations associated with anactivated electrode. Alternatively, acidic solution may be used.Alternatively, photolabile protecting groups on the amine may be usedsuch as those disclose in Fodor (cited previously).

In one or more embodiments, succinate moieties reacted to the aminoamidite moieties are referred to as reacted succinate moieties. In oneor more embodiments, the salt is a pyridinium salt as shown in FIG. 6,Compound B. Other salts of the succinate moieties may be used such astriethyl ammonium salt (Pierce Chemical Company), lutidine salt, orimidizole salt and salts having the form HN(R1R2R3)+, wherein R1, R2,and R3 are alkyl groups. HBTU/HOBT activation of the succinate moiety isa preferred embodiment. Other procedures to activate the succinate canbe used and include use of a carbodiimide such as N,N′-dicyclohexylcarbodiimide (DCC) or diisopropylcarbodiimide (DIC) both with or withoutN-hydrooxybenzotriazole (HOBt) or by forming a symmetrical anhydride.Use of other peptide coupling reagents such as2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate(TBTU), 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TNTU), O—(N-succinimidyl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TSTU),benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluorophasphate(BOP), benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphoniumhexafluorophosphate (PyBOP), bromo-tris-pyrrolidino-phosphoniumhexafluorophosphate (PyBroP), or 1,1′-carbonyl-diimidazole (CDI).

In one or more embodiments, oligomers are synthesized in situ usingelectrochemical synthesis. Electrochemical synthesis of DNA usesstandard phosphoramidite chemistry coupled with electrochemicaldeblocking of the protecting groups on the synthesized DNA for theaddition of each nucleotide contained in the oligonucleotide. Forattachment of the phosphoramidites, the microarray has hydroxyl groupsthat allow attachment of the first phosphoramidite. Electrochemicaldeblocking involves turning on an electrode to generate acidicconditions that are sufficient to remove the protecting group only atthe active electrode. Buffer in the solution used for deblocking andnatural diffusion prevents deblocking at non-activated electrodes.Removal of the protecting groups allows addition of the nextphosphoramidite.

Example 1

In this example, a CombiMatrix CustomArray™ 12 k microarray was used tosynthesize DNA attached to the microarray through a 10-T spacer and abase-cleavable sulfonyl linker. The microarray had approximately 12,000platinum electrodes on a solid surface having a porous reaction layer.Each electrode was electronically addressable via computer control. TheDNA was electrochemically synthesized in situ onto known locationsassociated with the electrodes on the microarray. The known locationswere on and within a porous reaction layer over the electrodes. Theporous reaction layer was composed of sucrose. The electrochemicalsynthesis used phosphoramidite chemistry coupled with electrochemicaldeblocking of the protecting groups on the synthesized DNA for theaddition of each subsequent nucleotide. For bonding of thephosphoramidites, the microarray had reactive hydroxyl groups providedby the sucrose. Electrochemical deblocking involved turning on anelectrode to generate acidic conditions at the electrode that weresufficient to remove the protecting group only at the active electrode.Buffer in the solution used for deblocking and natural diffusionprevented deblocking at non-activated electrodes. Removal of theprotecting group allowed addition of the next phosphoramidite.

The cleavable linker was at the end of the 10-T linker/spacer. Themicroarray was prepared by electrochemical synthesis of the 10-T linkeron all locations on the microarray. The final trityl on the 10-T linkerwas removed using electrochemically-generated acid. After synthesis ofthe linker and removal of the protecting groups on the linker, asolution having sulfonyl amidite was coupled to selected locations. Thesulfonyl amidite was 2-[2-(4,4′-dimethoxytrityloxy)ethylsulfonyl)ethyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite.The coupling solution comprised a 1:1 mixture of activator solution and100 mM sulfonyl amidite solution in acetonitrile. The solution was addedto a reaction chamber of the microarray immediately after mixing thecomponents. Care was taken to prevent water contamination during thecoupling step. The coupling reaction proceeded for 5 minutes. Thereaction chamber was evacuated, and an oxidation solution (Ox-T) wasinjected and allowed to react for 30 seconds to convert phosphorus IIIto phosphorus V. The reaction chamber was then cleaned thoroughly withacetonitrile.

A pool of DNA was synthesized onto the cleavable linkers. Aftersynthesis, the microarray was placed in a custom chamber and exposed toammonium hydroxide at 65° C. for 4-6 hours. The chamber was designed tobe able to withstand the pressures created by heating the solution up tothe temperature. During this step, the oligonucleotides were cleavedfrom the microarray and deprotected as the same time.

The microarray was placed on ice for about 10 minutes to allow theammonium hydroxide to cool to prevent the solution from spraying out ofthe chamber because of the relatively higher pressure inside thechamber. The ammonium hydroxide solution, which contained the targetoligonucleotides, was placed in a 65-microliter tube using a pipette.The ammonium hydroxide was removed using a SpeedVac® vacuum system at atemperature of about 65-85° C. until dry, which took about 30 minutes toan hour. The oligonucleotides were in the form of a pellet at the bottomof the tube. The oligonucleotides were resuspended in a Tris buffersolution and then cleaned using a Microspin® G-25 column obtained fromAmersham.

Example 2

Thirteen microarrays were prepared according to Example 1 but with someexceptions. First, the microarrays had the same oligonucleotide sequencesynthesized on each electrode rather than a pool of oligonucleotides.Additionally, six of the microarrays had the sulfonyl cleavable linker,and seven of the microarrays did not have the sulfonyl cleavable linker.

After synthesis of the oligonucleotides on the group of six microarrayshaving the cleavable linker, each of those microarrays was exposed to aconcentrated ammonium hydroxide solution for four hours at 65 degreesCelsius to remove the oligonucleotides. The oligonucleotides from eachmicroarray were recovered and amplified using PCR. The oligonucleotiderecovery was quantified using quantitative PCR, which used SYBR I as thefluorescent intercalating dye. The fluorescence intensity (FI) wasmonitored during PCR and plotted against the number of PCR cycles. TheFI for each reaction was normalized to the highest FI value. For eachreaction, the FI value at 50% of the maximum FI value was calculated foreach microarray, and the corresponding number of PCR cycles was obtainedby interpolation. The average number of PCR cycles to reach 50% FI valuefor the group six microarrays was about 11 cycles.

For the group of seven microarrays without the cleavable linker, each ofthose microarrays was exposed to different treatments in an attempt toremove the oligonucleotides for comparison to the cleavable linkermicroarrays. The treatments included 1% hydrogen peroxide, 1% hydrogenperoxide plus 0.2 molar sucrose, 0.1 molar hydrochloric acid, 0.4 molarhydrochloric acid, concentrated ammonium hydroxide, and methylamine. Theoligonucleotides from each microarray were recovered and amplified usingPCR. The fluorescence intensity (FI) was monitored during PCR andplotted against the number of PCR cycles. The FI was normalized to thehighest FI value. The FI value at 50% of the maximum FI value wascalculated for each microarray, and the corresponding number of PCRcycles was obtained by interpolation. The range of the number of PCRcycles to reach 50% FI value for the group seven microarrays was fromabout 27 cycles to about 35 cycles. Quantitative comparison of therecovery of oligonucleotides from microarrays with and without thecleavable linkers using a standard curve made from commerciallysynthesized oligonucleotides (of identical sequence) revealed that themicroarrays having the cleavable linker yielded an increased recovery ofapproximately six orders of magnitude over any other removal method weused.

Example 3

In this example, three different electrode microarrays were synthesizedwith each having different oligonucleotides ranging from 66 to 80 basepairs. Each microarray was prepared as in Example 1 except for thedifferent oligonucleotides. After synthesis, the microarrays wereexposed to concentrated ammonium hydroxide solution for four hours at 65degrees Celsius to remove the oligonucleotides. The oligonucleotidesfrom each microarray were recovered. The recovered oligonucleotides wereamplified using PCR. The amplified oligonucleotides were subjected togel electrophoresis. The gel was a 20% polyacrylamide gel. Theelectrophoresis conditions were 200 volts for 90 minutes. The expectedPCR product was 66 to 80 base pairs. FIG. 3 shows an image of theelectrophoresis gel. Oligonucleotides 321/322 were from the firstmicroarray. Oligonucleotides 323/324 were from the second microarray.Oligonucleotides 325/326 were from the third microarray. The laneshaving a positive sign are those where the oligonucleotides wereexpected to be located. The lanes having a negative sign are those wherethe oligonucleotides were not expected to be located. Thus, theolignucleotideos were cleaved from the microarray as expected.

Example 4

In this example, a CombiMatrix CustomArray™ 12K microarray was used tosynthesize oligonucleotides attached to the microarray through abase-cleavable linker. The microarray had approximately 12,000 platinumelectrodes on a solid surface having a porous reaction layer, whereineach electrode was electronically addressable via computer control. Theoligonucleotides were DNA and were synthesized in situ usingelectrochemical synthesis at locations associated with the electrodes onthe microarray. Electrochemical synthesis used standard phosphoramiditechemistry coupled with electrochemical deblocking of the protectinggroups on the synthesized DNA for the addition of each nucleotidecontained in the oligonucleotide. For attachment of thephosphoramidites, the microarray had organic reactive hydroxyl groupsthat allowed attachment of the first phosphoramidite. Electrochemicaldeblocking involved turning on an electrode to generate acidicconditions at the electrode that were sufficient to remove theprotecting group only at the active electrode. Buffer in the solutionused for deblocking and natural diffusion prevented deblocking atnon-activated electrodes. Removal of the protecting group allowedaddition of the next phosphoramidite.

Some electrodes were used as controls while some electrodes were used tosynthesize the oligonucleotides. At the non-control locations, a 15-unitdeoxythymidylate spacer was synthesized on the reactive hydroxyl groups.At some but not all non-control locations, an amine amidite obtainedfrom Glen Research was attached to the 15-unit spacer. The specificamine amidite was2-[2-(4-Monomethoxytrityl)aminoethoxy]ethyl-(2-cyanoethyl)-N,N-diisopropyl)-phosphoramidite,catalog number 10-1905-xx (5′-Amino-Modifier 5.) The amine amidite hadmonomethoxytrityl (MMT) protecting groups on the amine. The MMTprotecting groups were removed using electrochemical generation of acidby activating selected electrodes.

After removal of the MMT protecting groups, the amine was reacted to aT-succinate to form an amide linkage between the amine groups and thesuccinate. The specific T-succinate used was5′-dimethyloxytrityl-thymidine-3′-O-succinate (pyridium salt) obtainedfrom Transgenomic. (Alternatively, A, C, G, succinates could have beenused.) The solution to attach the T-succinate to the amine was made byadding 330 milligrams of T-succinate, 150 milligrams ofO-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluorophosphate(HBTU), and 60 milligrams of N-hydroxybenztriazole (HOBT) to onemilliliter dimethyl formamide(DMF). To this solution, 225 microliters ofdiisopropylethylamine (DIEPA) was added and the resulting mixture wasvortexed to dissolve the material (total mixing time 5-10 min) prior touse.

To attach the T-succinate, the microarray was placed in a manifold,rinsed with anhydrous DMF, and exposed to one-half of the T-succinatecoupling mixture for one hour at room temperature. The microarray waswashed in the manifold using different solvents successively as follows:5 milliliters of DMF, 5 milliliters of methylene chloride, and 5milliliter of DMF. The microarray was exposed to the second half of thecoupling mixture for one hour at room temperature. After the completionof the second exposure to the T-succinate reaction mixture, themicroarray was washed again using the same washing as above followed bymethylene chloride (5 ml) and a stream of ethanol from a squirt bottle.After washing, the microarray was ready for electrochemical synthesis.Synthesis was done on a CombiMatrix bench top synthesizer, whereinoligonucleotides of three different lengths (37, 42, and 47 bp) weresynthesized.

After the completion of electrochemical synthesis, the syntheticoligonucleotides on the microarray were deprotected and cleaved byexposure to concentrated ammonium hydroxide in a pressurized cell at 65°C. The concentration of ammonium hydroxide was 28-30%. During thisdeprotection step, the cleavable succinate linkage was cleaved thusreleasing the synthetic oligonucleotides. The oligonucleotides wereisolated by evaporating the ammonia solution and were subjected toamplification using polymerase chain reaction (PCR). Theoligonucleotides could be amplified with one set of PCR primers due tothe presence of primer amplification sites at the ends of theoligonucleotides. The oligonucleotides were dissolved in 75 microlitersof Tris buffer at 95° C. for 5 minutes.

To test if the nucleotides were released, a series of PCR reactions wereperformed to determine if the oligonucleotides were present in solution.PCR reaction products were run on a non-denaturing polyacrylamide gel(20%) for 100 minutes at 200 volts. When the separation of the PCRproducts was complete, the gel was stained with SYBR green II dye tovisualize the PCR product as shown in FIG. 7. Separation of the PCRproduct by gel electrophoresis revealed that all three products (37, 42,and 47 bp) were present in approximately equal amounts and ran at thecalculated molecular weight. The novel linker allowed for the release ofoligonucleotides from the microarray surface.

At some locations on the array, the oligonucleotides synthesized wereattached without the cleavable linker. Thus, oligonucleotides attachedwithout the cleavable linker would be expected to remain on themicroarray after the ammonium hydroxide reaction. To determine whetherthere were oligonucleotides remaining on the microarray after theammonium hydroxide reaction, the microarray was exposed to complementaryoligonucleotides having a fluorescent label. FIG. 4 shows an image of aportion of the microarray after exposure to the fluorescently labeledoligonucleotide. There are four different areas, A, B, C, and D, shownin the figure. In areas A, B, and C, oligos were synthesized with andwithout the cleavable linker. As can be seen in the figure, themicroarray locations having the cleavable linker between theoligonucleotide and the microarray are completely dark or are mostlydark indicating little or no DNA remains after cleaving. In contrast,those locations that did not have the cleavable linker between theoligonucleotide and the microarray are brighter, which indicates thatthe oligonucleotide remained on the microarray. In area D, someelectrodes had cleavable linker while some did not; however, no oligowas synthesized so that the entire area appears dark.

In an embodiment of the invention, a process of forming a microarrayhaving base cleavable phosphoramidite linkers comprises providing amicroarray having a surface with a plurality of known locations on thesurface, wherein each location has a plurality of hydroxyl groups,wherein the density of the known locations is greater than approximately100 per square centimeter on the surface and bonding a plurality of basecleavable phosphoramidite linkers to the plurality of hydroxyl groupsdirectly or by using an intermediate chemical moiety attached to thehydroxyl groups to form a plurality of cleavable linkers at theplurality of known locations, wherein the cleavable linkers each have alinker hydroxyl group and a base-labile cleaving site, wherein thelinker hydroxyl group is protected by a protecting group and thebase-labile cleaving site is an ether linkage.

In an embodiment of the invention, a process of forming a microarrayhaving base cleavable phosphoramidite linkers comprises providing amicroarray having a surface with a plurality of known locations on thesurface, wherein each location has a plurality of hydroxyl groups,wherein the density of the known locations is greater than approximately100 per square centimeter on the surface and bonding a plurality of basecleavable phosphoramidite linkers to the plurality of hydroxyl groupsdirectly or by using an intermediate chemical moiety attached to thehydroxyl groups to form a plurality of cleavable linkers at theplurality of known locations, wherein the cleavable linkers each have alinker hydroxyl group and a base-labile cleaving site, wherein thelinker hydroxyl group is protected by a protecting group and thebase-labile cleaving site is an ether linkage, further comprisingsynthesizing oligomers onto the linker hydroxyl groups to provide amicroarray of oligomers, wherein the protecting group is removed fromthe linker hydroxyl groups before synthesizing the oligomers, whereinthe oligomers at the known locations, as between different knownlocations, are different or the same.

In an embodiment of the invention, a process of forming a microarrayhaving base cleavable phosphoramidite linkers comprises providing amicroarray having a surface with a plurality of known locations on thesurface, wherein each location has a plurality of hydroxyl groups,wherein the density of the known locations is greater than approximately100 per square centimeter on the surface and bonding a plurality of basecleavable phosphoramidite linkers to the plurality of hydroxyl groupsdirectly or by using an intermediate chemical moiety attached to thehydroxyl groups to form a plurality of cleavable linkers at theplurality of known locations, wherein the cleavable linkers each have alinker hydroxyl group and a base-labile cleaving site, wherein thelinker hydroxyl group is protected by a protecting group and thebase-labile cleaving site is an ether linkage, further comprisingsynthesizing oligomers onto the linker hydroxyl groups to provide amicroarray of oligomers, wherein the protecting group is removed fromthe linker hydroxyl groups before synthesizing the oligomers, whereinthe oligomers at the known locations, as between different knownlocations, are different or the same, and cleaving at the base-labilecleaving site the oligomers from the surface using a cleaving base toprovide a pool of cleaved oligomers.

In an embodiment of the invention, a process of forming a microarrayhaving base cleavable phosphoramidite linkers comprises providing amicroarray having a surface with a plurality of known locations on thesurface, wherein each location has a plurality of hydroxyl groups,wherein the density of the known locations is greater than approximately100 per square centimeter on the surface and bonding a plurality of basecleavable phosphoramidite linkers to the plurality of hydroxyl groupsdirectly or by using an intermediate chemical moiety attached to thehydroxyl groups to form a plurality of cleavable linkers at theplurality of known locations, wherein the cleavable linkers each have alinker hydroxyl group and a base-labile cleaving site, wherein thelinker hydroxyl group is protected by a protecting group and thebase-labile cleaving site is an ether linkage, further comprisingsynthesizing oligomers onto the linker hydroxyl groups to provide amicroarray of oligomers, wherein the protecting group is removed fromthe linker hydroxyl groups before synthesizing the oligomers, whereinthe oligomers at the known locations, as between different knownlocations, are different or the same, and cleaving at the base-labilecleaving site the oligomers from the surface using a cleaving base toprovide a pool of cleaved oligomers, wherein the oligomers are selectedfrom the group consisting of DNA, RNA, and polypeptides, andcombinations thereof.

In an embodiment of the invention, a process of forming a microarrayhaving base cleavable phosphoramidite linkers comprises providing amicroarray having a surface with a plurality of known locations on thesurface, wherein each location has a plurality of hydroxyl groups,wherein the density of the known locations is greater than approximately100 per square centimeter on the surface and bonding a plurality of basecleavable phosphoramidite linkers to the plurality of hydroxyl groupsdirectly or by using an intermediate chemical moiety attached to thehydroxyl groups to form a plurality of cleavable linkers at theplurality of known locations, wherein the cleavable linkers each have alinker hydroxyl group and a base-labile cleaving site, wherein thelinker hydroxyl group is protected by a protecting group and thebase-labile cleaving site is an ether linkage, further comprisingsynthesizing oligomers onto the linker hydroxyl groups to provide amicroarray of oligomers, wherein the protecting group is removed fromthe linker hydroxyl groups before synthesizing the oligomers, whereinthe oligomers at the known locations, as between different knownlocations, are different or the same, and cleaving at the base-labilecleaving site the oligomers from the surface using a cleaving base toprovide a pool of cleaved oligomers, wherein the cleaving base isselected from the group consisting of chemical base, ammonium hydroxide,electrochemically generated base, sodium hydroxide, potassium hydroxide,methylamine, and ethylamine and combinations thereof.

In an embodiment of the invention, a process of forming a microarrayhaving base cleavable phosphoramidite linkers comprises providing amicroarray having a surface with a plurality of known locations on thesurface, wherein each location has a plurality of hydroxyl groups,wherein the density of the known locations is greater than approximately100 per square centimeter on the surface and bonding a plurality of basecleavable phosphoramidite linkers to the plurality of hydroxyl groupsdirectly or by using an intermediate chemical moiety attached to thehydroxyl groups to form a plurality of cleavable linkers at theplurality of known locations, wherein the cleavable linkers each have alinker hydroxyl group and a base-labile cleaving site, wherein thelinker hydroxyl group is protected by a protecting group and thebase-labile cleaving site is an ether linkage, further comprisingsynthesizing oligomers onto the linker hydroxyl groups to provide amicroarray of oligomers, wherein the protecting group is removed fromthe linker hydroxyl groups before synthesizing the oligomers, whereinthe oligomers at the known locations, as between different knownlocations, are different or the same, and cleaving at the base-labilecleaving site the oligomers from the surface using a cleaving base toprovide a pool of cleaved oligomers, wherein the oligomers aresynthesized in situ using electrochemical synthesis.

In an embodiment of the invention, a process of forming a microarrayhaving base cleavable phosphoramidite linkers comprises providing amicroarray having a surface with a plurality of known locations on thesurface, wherein each location has a plurality of hydroxyl groups,wherein the density of the known locations is greater than approximately100 per square centimeter on the surface and bonding a plurality of basecleavable phosphoramidite linkers to the plurality of hydroxyl groupsdirectly or by using an intermediate chemical moiety attached to thehydroxyl groups to form a plurality of cleavable linkers at theplurality of known locations, wherein the cleavable linkers each have alinker hydroxyl group and a base-labile cleaving site, wherein thelinker hydroxyl group is protected by a protecting group and thebase-labile cleaving site is an ether linkage, further comprisingsynthesizing oligomers onto the linker hydroxyl groups to provide amicroarray of oligomers, wherein the protecting group is removed fromthe linker hydroxyl groups before synthesizing the oligomers, whereinthe oligomers at the known locations, as between different knownlocations, are different or the same, and cleaving at the base-labilecleaving site the oligomers from the surface using a cleaving base toprovide a pool of cleaved oligomers, wherein the oligomers aresynthesized in situ by a method selected from the group consisting of(i) printing reagents via ink jet or other printing technology and usingregular phosphoramidite chemistry, (ii) maskless photo-generated acidcontrolled synthesis and using regular phosphoramidite chemistry, (iii)mask-directed parallel synthesis using photo-cleavage of photolabileprotecting groups, and (iv) maskless parallel synthesis usingphoto-cleavage of photolabile protecting groups and digitalphotolithography.

In an embodiment of the invention, a pool of oligomers producedaccording to the process of forming a microarray having base cleavablephosphoramidite linkers comprises providing a microarray having asurface with a plurality of known locations on the surface, wherein eachlocation has a plurality of hydroxyl groups, wherein the density of theknown locations is greater than approximately 100 per square centimeteron the surface and bonding a plurality of base cleavable phosphoramiditelinkers to the plurality of hydroxyl groups directly or by using anintermediate chemical moiety attached to the hydroxyl groups to form aplurality of cleavable linkers at the plurality of known locations,wherein the cleavable linkers each have a linker hydroxyl group and abase-labile cleaving site, wherein the linker hydroxyl group isprotected by a protecting group and the base-labile cleaving site is anether linkage, further comprising synthesizing oligomers onto the linkerhydroxyl groups to provide a microarray of oligomers, wherein theprotecting group is removed from the linker hydroxyl groups beforesynthesizing the oligomers, wherein the oligomers at the knownlocations, as between different known locations, are different or thesame, and cleaving at the base-labile cleaving site the oligomers fromthe surface using a cleaving base to provide a pool of cleavedoligomers, wherein the oligomers are oligonucleotides having a 3′phosphate, wherein the pool comprises more than approximately 100different oligonucleotides.

In an embodiment of the invention, a pool of oligomers producedaccording to the process of forming a microarray having base cleavablephosphoramidite linkers comprises providing a microarray having asurface with a plurality of known locations on the surface, wherein eachlocation has a plurality of hydroxyl groups, wherein the density of theknown locations is greater than approximately 100 per square centimeteron the surface and bonding a plurality of base cleavable phosphoramiditelinkers to the plurality of hydroxyl groups directly or by using anintermediate chemical moiety attached to the hydroxyl groups to form aplurality of cleavable linkers at the plurality of known locations,wherein the cleavable linkers each have a linker hydroxyl group and abase-labile cleaving site, wherein the linker hydroxyl group isprotected by a protecting group and the base-labile cleaving site is anether linkage, further comprising synthesizing oligomers onto the linkerhydroxyl groups to provide a microarray of oligomers, wherein theprotecting group is removed from the linker hydroxyl groups beforesynthesizing the oligomers, wherein the oligomers at the knownlocations, as between different known locations, are different or thesame, and cleaving at the base-labile cleaving site the oligomers fromthe surface using a cleaving base to provide a pool of cleavedoligomers, wherein the oligomers are oligonucleotides having a 3′hydroxyl, wherein the pool comprises more than approximately 100different oligonucleotides.

In an embodiment of the invention, a process of forming a microarrayhaving base cleavable phosphoramidite linkers comprises providing amicroarray having a surface with a plurality of known locations on thesurface, wherein each location has a plurality of hydroxyl groups,wherein the density of the known locations is greater than approximately100 per square centimeter on the surface and bonding a plurality of basecleavable phosphoramidite linkers to the plurality of hydroxyl groupsdirectly or by using an intermediate chemical moiety attached to thehydroxyl groups to form a plurality of cleavable linkers at theplurality of known locations, wherein the cleavable linkers each have alinker hydroxyl group and a base-labile cleaving site, wherein thelinker hydroxyl group is protected by a protecting group and thebase-labile cleaving site is an ether linkage, further comprisingsynthesizing oligomers onto the linker hydroxyl groups to provide amicroarray of oligomers, wherein the protecting group is removed fromthe linker hydroxyl groups before synthesizing the oligomers, whereinthe oligomers at the known locations, as between different knownlocations, are different or the same, and cleaving at the base-labilecleaving site the oligomers from the surface using a cleaving base toprovide a pool of cleaved oligomers, wherein the surface has electrodesand each of the known locations are associated with one of theelectrodes, wherein the electrodes are electronically addressable.

In an embodiment of the invention, a pool of oligomers producedaccording to the process of forming a microarray having base cleavablephosphoramidite linkers comprises providing a microarray having asurface with a plurality of known locations on the surface, wherein eachlocation has a plurality of hydroxyl groups, wherein the density of theknown locations is greater than approximately 100 per square centimeteron the surface and bonding a plurality of base cleavable phosphoramiditelinkers to the plurality of hydroxyl groups directly or by using anintermediate chemical moiety attached to the hydroxyl groups to form aplurality of cleavable linkers at the plurality of known locations,wherein the cleavable linkers each have a linker hydroxyl group and abase-labile cleaving site, wherein the linker hydroxyl group isprotected by a protecting group and the base-labile cleaving site is anether linkage, further comprising synthesizing oligomers onto the linkerhydroxyl groups to provide a microarray of oligomers, wherein theprotecting group is removed from the linker hydroxyl groups beforesynthesizing the oligomers, wherein the oligomers at the knownlocations, as between different known locations, are different or thesame, and cleaving at the base-labile cleaving site the oligomers fromthe surface using a cleaving base to provide a pool of cleavedoligomers, wherein the oligomers are oligonucleotides having a 3′hydroxyl, wherein the pool comprises more than approximately 100different oligonucleotides, wherein the known locations are on the samesurface as the electrodes, on an opposing surface to the electrodes, oron an overlayer over the electrodes.

In an embodiment of the invention, a microarray having base cleavablelinkers comprises a microarray having a surface with a plurality ofknown locations on the surface, wherein each location has a plurality ofhydroxyl groups, wherein the density of the known locations is greaterthan approximately 100 per square centimeter on the surface and aplurality of base cleavable linkers bonded to the plurality of hydroxylgroups to form a plurality of cleavable linkers at the plurality ofknown locations, wherein the cleavable linkers each have a linkerhydroxyl group and a base-labile cleaving site.

We claim:
 1. A process of forming a pool of oligomers comprising: (a)providing a microarray having a surface with a plurality of knownlocations on the surface, wherein each location has a plurality ofhydroxyl surface bound species, wherein a plurality of hydroxyl surfacebound species are bound to a plurality of electronically addressableelectrodes; (b) bonding a plurality of monomers at the plurality ofknown locations on the surface to generate a spacer bearing subsequenthydroxyl surface bound species; (c) bonding a plurality of amine amiditelinkers including a phosphorous moiety which forms a phosphorous-oxygenbond with the subsequent hydroxyl surface bound species to form aplurality of protected amine surface bound species, wherein theplurality of protected amine surface bound species include an acidlabile protecting group; (d) generating acidic conditions to remove theprotecting groups of step (c) and reacting a protected succinatenucleoside moiety therewith to form a plurality of protected succinatenucleoside bound species, wherein the plurality of protected succinatenucleoside bound species include an acid-labile group protecting its5′OH; (e) turning on one or more of the plurality of electronicallyaddressable electrodes to generate acidic conditions to removeprotecting groups from a plurality of succinate nucleoside bound speciesin one or more known first locations selected from the plurality ofknown locations; (f) reacting a first protected monomer with at leastthe known first locations of step (e) to form a plurality of firstprotected monomer bound species, wherein the plurality of firstprotected monomer bound species include an acid-labile 5′ protectinggroup; (g) turning on one or more of the plurality of electronicallyaddressable electrodes to generate acidic conditions to remove theprotecting groups from a second plurality of succinate nucleoside boundspecies in one or more known second locations selected from theplurality of known locations; (h) reacting a second protected monomerwith the one or more known second locations to form a plurality ofsecond protected monomer bound species, wherein the plurality of secondprotected monomer bound species include an acid-labile 5′ protectinggroup; (i) turning on one or more of the plurality of electronicallyaddressable electrodes to generate acidic conditions to remove theacid-labile 5′ protecting group in one or more known third locationsselected from the plurality of known locations; (j) reacting a thirdprotected monomer at the one or more known third locations, wherein thethird protected monomer includes an acid-labile 5′ protecting group; (k)repeating steps (i) through (j) to generate a plurality of oligomers,wherein the plurality of oligomers differ as to their sequence asbetween different known locations; and (l) forming a pool of oligomersby introducing a cleaving base to cleave the succinate base-labilecleavage site of the plurality of oligomers.
 2. The process of claim 1,wherein the pool of oligomers are selected from the group consisting ofDNA, RNA and combinations thereof.
 3. The process of claim 1, whereinthe cleaving base is one or more compounds selected from the groupconsisting of chemical base, ammonium hydroxide, electrochemicallygenerated base, sodium hydroxide, potassium hydroxide, methylamine, andethylamine.
 4. The process of claim 1, wherein the oligomers aresynthesized in situ using electrochemical synthesis.
 5. The process ofclaim 1, wherein the oligomers are synthesized in situ by a methodselected from the group consisting of (i) printing reagents via ink jetor other printing technology and using regular phosphoramiditechemistry, (ii) maskless photo-generated acid controlled synthesis andusing regular phosphoramidite chemistry, (iii) mask-directed parallelsynthesis using photo-cleavage of photolabile protecting groups, and(iv) maskless parallel synthesis using photo-cleavage of photolabileprotecting groups and digital photolithography.
 6. The process of claim1, wherein the pool of oligomers comprises more than approximately 100different oligonucleotides.
 7. The process of claim 1, wherein the firstprotected monomer includes a base-labile protecting group on thenucleobase.
 8. The process of claim 1, further comprising wherein thecleaving base is electrochemically generated.
 9. The process of claim 8,further comprising adding one or more compounds selected from the groupconsisting of ammonium hydroxide, sodium hydroxide, potassium hydroxide,methylamine, and ethylamine.
 10. The process of claim 1, wherein anoligomer is added using electrochemical synthesis.
 11. The process ofclaim 1, where the pool of oligomers have a free hydroxyl at the 3′position.
 12. A process of forming a pool of oligomers comprising: (a)providing a microarray having a surface with a plurality of knownlocations on the surface, where each location has a first plurality ofhydroxyl surface bound species, where the first plurality of hydroxylsurface bound species are bound to a plurality of electronicallyaddressable electrodes; (b) bonding a plurality of monomers at theplurality of known locations on the surface to generate a spacerproviding a second plurality of hydroxyl surface bound species; (c)bonding a plurality of amine amidite linkers including a phosphorousmoiety which forms a phosphorous-oxygen bond with the second pluralityof hydroxyl surface bound species to form a plurality of protected aminosurface bound species, where the plurality of protected amino surfacebound species include an acid labile protecting group; (d) turning onone or more of the plurality of electronically addressable electrodes togenerate acidic conditions to remove the protecting groups from aplurality of amino bound species in at least one of the first locationsof step (b) selected from the plurality of known locations; (e) reactinga first protected succinate nucleoside moiety with the plurality ofamino bound species of step (d) to form a first plurality of protectedsuccinate nucleoside bound species, where the first plurality ofprotected succinate nucleoside bound species includes an acid-labile 5′protecting group; (f) turning on one or more of the plurality ofelectronically addressable electrodes to generate acidic conditions toremove the protecting groups from a plurality of amino bound species inat least one location of step (e) selected from the plurality of knownlocations; (g) reacting a second protected succinate nucleoside moietywith the plurality of amino bound species of step (c) to form a secondplurality of protected succinate nucleoside bound species, where thesecond plurality of protected succinate nucleoside bound species includean acid-labile 5′ protecting group, where the second plurality ofprotected succinate nucleoside bound species includes a succinatebase-labile cleavage site; (h) turning on one or more of a plurality ofelectronically addressable electrodes to generate acidic conditions toremove the protecting groups from a plurality of protected succinatenucleoside bound species in at least one location of step (g) selectedfrom the plurality of known locations; (i) bonding a plurality ofprotected monomers including a phosphorous moiety in at least onelocation of step (h), where the plurality of protected monomers includean acid-labile 5′ protecting group; (j) turning on one or more of theplurality of electronically addressable electrodes to generate acidicconditions to remove the protecting groups in at least one location ofstep (i) selected from the plurality of known locations and bonding aplurality of protected monomers including a phosphorous moiety at theone or more known fourth locations, where the plurality of protectedmonomers include an acid-labile 5′ protecting group; (k) repeating steps(h) through (j) to generate a plurality of oligomers, where theplurality of oligomers differ as to their sequence as between differentknown locations; and (l) forming a pool of oligomers by introducing acleaving base to cleave the succinate base-labile cleavage site of theplurality of oligomers.
 13. The process of claim 12, where the pluralityof protected monomers includes a base-labile protecting group on thenucleobase.
 14. The process of claim 12, where the pool of oligomers areselected from the group consisting of DNA, RNA, and combinationsthereof.
 15. The process of claim 12, further comprising where thecleaving base is electrochemically generated.
 16. The process of claim15, further comprising adding one or more compounds selected from thegroup consisting of ammonium hydroxide, sodium hydroxide, potassiumhydroxide, methylamine, and ethylamine.
 17. The process of claim 12,where the cleaving base is one or more compounds selected from the groupconsisting of ammonium hydroxide, sodium hydroxide, potassium hydroxide,methylamine, and ethylamine.
 18. The process of claim 12, where anoligomer is added using electrochemical synthesis.
 19. The process ofclaim 12, where the oligomers are synthesized in situ by a methodselected from the group consisting of (i) printing reagents via ink jetor other printing technology and using regular phosphoramiditechemistry, (ii) maskless photo-generated acid controlled synthesis andusing regular phosphoramidite chemistry, (iii) mask-directed parallelsynthesis using photo-cleavage of photolabile protecting groups, and(iv) maskless parallel synthesis using photo-cleavage of photolabileprotecting groups and digital photolithography.
 20. The process of claim12, where the pool of oligomers comprises more than approximately 100different oligonucleotides.
 21. The process of claim 12, where the poolof oligomers have a free hydroxyl at the 3′ position.